Heat shock factor
The developmental and heat-shock-induced expression of two small heat-shock proteins (Hsp23 and Hsp27) was investigated during spermatogenesis in Drosophila melanogaster. Both of these Hsps are expressed in unstressed and stressed male gonads as shown by immunoblotting. Immunostaining of whole-mount organs and thin sections of testes showed that an anti-Hsp23 antibody specifically decorated cells of the somatic lineage, such as the cyst cells and the epithelial cells of the testis and of the seminal vesicle. Hsp27 is expressed in some somatic cells (cyst cells and epithelial cells of the accessory glands) and, in addition, is also visible in the maturing spermatocytes of the germline. The same cell-specific pattern of expression is observed after heat shock, and cells which do not express Hsp23 and Hsp27 in the absence of stress are similarly unable to mount a heat shock response for these s-Hsps. However other Hsps such as Hsp70 and Hsp22 are induced under heat-shock conditions in testes. Actinomycin D prevents the heat-induced accumulation of these Hsps indicating that the induction of Hsps is regulated at the transcriptional level. The heat shock transcriptional factor of Drosophila (HSF), present in significantly lower amount in testes when compared to other tissues such as the head, is shown to be required for the heat activation of Hsp22 and Hsp70. Immunostaining revealed that HSF expression is restricted to specific cells such as cyst cells, epithelial pigment cells, spermatogonia and spermatids but not the primary spermatocytes. These data show that the expression and induction of the different small Hsps is regulated in a cell-specific manner under both normal and heat shock conditions and suggest that factors other than the HSF are involved in this regulation in male gonads (Michaud, 1997).
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. The interaction of the 5-bp unit with HSFs of Drosophila and Saccharomyces has been investigated. Mutations within the conserved, central trinucleotide GAA reduces 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 indicates 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. Thus HSF recognizes the HSE primarily through contacts within the major groove of the DNA helix. Based on these observations and a re-evaluation of the base frequencies and criteria for consensus sequence assignment, it is proposed that the sequence AGAAN more accurately represents the consensus HSE motif (Fernandes, 1993).
Cooperative binding of HSF requires the close proximity of HSEs, rather than their helical alignment. Two or more trimeric HSEs organized as contiguous 5 bp units show much higher levels of cooperativity than multiple but separated HSEs. 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 (Fernandes, 1995).
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. Similar to deletion of HSEII, insertion in the spacer between the HSEs of 1 to 5 or 11 to 14 nucleotides (nt) reduces promoter activity to about 10% of that of the unaltered promoter. In contrast, HSEII is capable of contributing to promoter activity when the spacer is 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 is ineffective when its distance to HSEI is increased by more than 18 nt. In vitro, HSEII is a weak binding site for heat shock transcription factor, while HSEI is a strong binding site; 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 reveal cooperative binding of purified, recombinant HSF to HSEII with a similar periodicity as observed in vivo. Changing the distance between TATA and the HSEs does not produce promoter periodicity, indicating that stereoalignment of these elements is not important (Amin, 1994).
The parasitic helminth Schistosoma contains HSF activity that correlates with the pattern of hsp70 mRNA levels at different developmental stages in this organism. HSF activity revealed in extracts of Schistosoma mansoni (Sm) was further analyzed by competition experiments and compared with the well characterized HSF of Drosophila (Dm). The interactions of HSF in Sm extracts (SmHSF) and HSF of Dm (DmHSF) with 32P-labeled HSE probes, with and without unlabeled competitor DNA probes (HSE-related oligos), were analyzed by gel retardation assay. The binding and inhibition studies demonstrate that SmHSF and DmHSF differ in HSE sequence recognition: an array of three nGAAn inverted repeats according to the ideal consensus sequence (nGAAnnTTCnnGAAn) is recognized by DmHSF, but not by SmHSF. In the schistosome, binding is attained only when the third pentamer is a variant, composed of nGTAn instead of nGAAn. The presence of this variant in the promoter of the hsp70 gene of the parasite suggests coevolution of the variant sequence together with the SmHSF, which interacts efficiently with the variant, but not with the ideal HSE sequence. Further inhibition studies reveal additional differences between SmHSF and DmHSF in recognition of the first and second nGAAn pentamers of HSE. By analogy to other systems of ligand-protein interactions, it has been proposed that the complementarity between the HSE ligand and the HSF protein is higher in SmHSF, as compared to DmHSF (Levy-Holtzman, 1995).
The chromatin structure of the Drosophila hsp26 promoter is characterized by two DNAase hypersensitive (DH) sites harboring regulatory elements. Proximal and distal DH sites are separated by a positioned nucleosome. Both DH sites are readily reconstituted from extract components. They are separated by a nucleosome which is less strictly positioned than its in vivo counterpart. The interaction of GAGA factor (Trithorax-like) and Heat shock factor with their binding sites in chromatin occurs in two modes. Their interaction with binding sites in the nucleosome free regions does not require ATP. In the presence of ATP both factors also interact with nucleosomal binding sites, causing nucleosome rearrangements and a refinement of nucleosome positions. While chromatin remodeling upon transcription factor interaction has previously been interpreted to involve nucleosome disruption, the data suggest instead that energy-dependent nucleosome sliding is the main principle of chromatin reorganization (Wall, 1995).
Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes was studied to provide a framework for evaluating the mechanisms of activation and repression of transcription. RNA polymerase transcription at the 5' end of the hsp70 gene can be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase can already be detected near the 3' end of the gene. A similar rapid induction is found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene is found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. The in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene is approximately 1.2 kb/min (O'Brien, 1993).
Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock, which strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, it has been found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties (Karunanithi, 1999).
Since purified GAGA factor and TFIID interact similarly with the hsp70 and histone H3 promoters, the architecture of the endogenous H3 promoter has been analyzed to determine what interactions might be needed to establish a potentiated state containing a pause as seen in HSP70 promoter. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase as seen in the HSP70 promoter 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 (Weber, 1995).
Three promoter sequences 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 to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, the in vivo occupancy of the TATA elements were examined in the transgenic promoters. TATA occupancy correlates with HSF binding for some promoters. However, in all cases HSF accessibility correlates with the presence of paused RNA polymerase II. Thus, a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase II (Shopland, 1995).
There are two modes of regulation of the hsp26 promoter: one for promoters before heat shock, and another for promoters after heat shock. Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). Transcription in extracts from unstressed embryos relies solely on GAGA elements that efficiently counteract repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depends only a little on GAGA elements, relying mainly on functional HSEs. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo (Sandaltzopoulos, 1995).
Transcription by RNA polymerase II is highly regulated at the level of initiation and elongation. Well-documented transcription activation mechanisms, such as the recruitment of TFIID and TFIIB, control the early phases of preinitiation complex formation. The heat shock genes provide an example of transcriptional regulation at a later step: in nuclei, TFIID can be detected at the TATA box prior to heat induction. Using cell-free systems for chromatin reconstitution and transcription, the mechanisms by which heat shock factor (HSF) increases transcription of heat shock genes in chromatin have been analyzed. HSF affects transcription of naked DNA templates in multiple ways: (1) by speeding up the rate of preinitiation complex formation (PIC), (2) by increasing the number of productive templates, and (3) by increasing the reinitiation rate. Under the more physiological conditions of potentiated chromatin templates, HSF affects only the reinitiation rate (Sandaltzopoulos, 1998)
In vivo, the first initiation of hsp26 transcription occurs in the absence of heat shock under conditions where HSF is unable to bind promoters with high specificity. Under such conditions, productive transcription from the hsp26 promoter requires the efficient release of the pausing polymerase as well as the recruitment of the transcription machinery to the TFIID-TFIIA complex for reinitiation. Conceivably, HSF may contact component(s) of the transcription machinery other than TFIID-TFIIA or a putative polymerase holo-enzyme complex. Such a recruitment would speed up the formation of the first PIC and subsequent reinitiation events and might also increase the template usage. In crude transcription systems an equilibrium between the formation of productive and nonproductive preinitiation complexes exists, brought about by competition between negative and positive regulators. HSF might increase the fraction of active templates by shifting this equilibrium towards the formation of productive complexes. It is, however, also possible that HSF increases the affinity of the committed complex for TFIIB by altering the conformation of a ternary TFIIA-TFIID-TATA complex (Sandaltzopoulos, 1998).
Reinitiation by newly recruited machinery is only possible if the previous polymerase has cleared the promoter. In heat shock genes, promoter clearance is a limiting step that can be overcome by activated HSF. Promoter clearance involves the phosphorylation of the polymerase CTD by the TFIIH kinase, a process that is integrated by TFIIE: TFIIE associates selectively with the hypophosphorylated form of the polymerase and stimulates the TFIIH kinase at a late step in PIC assembly. In vivo the paused polymerase at the hsp26 promoter should be in the elongation phase, but it has an unphosphorylated CTD. Since HSF induction leads to the release of this polymerase, it will be interesting to investigate whether HSF re-recruits TFIIE and TFIIH, as has been suggested, or an as yet unidentified heat shock-specific CTD kinase. Activator-dependent reinitiation of transcription, obviating the slow assembly of the TFIID-TFIIA complex on a promoter, may be especially crucial for genes requiring a fast response to inducers (Sandaltzopoulos, 1998).
The transition of heat shock factor to the induced state is accompanied by a chromosomal redistribution of HSF to the heat-shock puff sites. Over 150
additional chromosomal sites also accumulate HSF, including developmental loci that are repressed during heat shock. These findings suggest an unforeseen role for HSF as a repressor of normal gene activity during heat stress (Westwood, 1991).
To decipher the mechanistic roles of Mediator proteins in regulating
developmental specific gene expression and compare them
to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing
Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal
transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement
for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by
promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates
promoter-specific activation signals to the basal transcription machinery (Park, 2001).
Previous studies in yeast and human cells have suggested
that transcriptional activator proteins interact with Mediator
complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as
TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been
suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes
with the VP16 protein were examined. After incubation of nuclear extracts with an
excess of GST fusion protein beads containing either wild-type or
mutant (Delta456FP442) VP16 activation domain, the supernatants were
analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating
with GST-VP16 but not with GST-VP16Delta456FP442. However, the amounts
of dGCN5, dTAFII40, dTAFII250, and dTBP in the
extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID
and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the
amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most
strongly bound to and most readily recruited to the activation domain (Park, 2001).
In addition to the model VP16 activator derived from herpesvirus,
dMediator interacts with Drosophila transcriptional
activators Dorsal and heat shock factor (dHSF). When dMediator complex
was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads
even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein
G-agarose beads through anti-dSOH1 Ab and the binding of
diverse 35S-labeled Drosophila transcription
factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained
specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).
To evaluate the requirement of dMediator for activated transcription in
response to the Drosophila activator proteins that interact
with dMediator, the ability of dMediator-deficient nuclear
extracts to support transcriptional activation by the Dorsal and
Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to
mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level
of transcriptional activation is reduced significantly (five- and
three-fold activations, respectively) in nuclear extract that has been
depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by
the sequence-specific transcription factor Even-skipped (Park, 2001).
dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer
elements. However, the requirement of TAFs, or at least
dTAFII250, in activated transcription appears to be
redundant in the in vitro transcription system used and affected by
such factors as the core promoter organization or nucleosomal structure
of transcriptional templates. Several TAF components in the TFIID
complex indeed have biochemical activities and structural motifs
adequate for the recognition of specialized settings of transcription
templates. For example, certain TAFs recognize the Inr and DPE
sequences located in many Drosophila core promoters and
increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAFII250, which has a HAT catalytic activity and also possesses a
histone octamer-like module comprising the histone H2B-, H3-, and
H4-like TAFs. Although not experimentally
demonstrated, these TAFs may have some roles in the transcriptional
regulation of nucleosomal templates (Park, 2001).
The sequence-specific transcription factors which interact physically with
dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and
Fushi-tarazu. These factors contain different types of
activation domains (acidic and glutamine-rich domains). Most of these
transcription factors have been shown to activate transcription either
constitutively or inducibly. It is noteworthy that dHSF interacts with
and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170),
suggests that some function of Mediator is required for HSF-mediated
transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is
conserved between yeast and Drosophila,
transcriptional activation by HSF might utilize the conserved Rgr1
components of the Mediator complexes (Park, 2001).
Although some human Mediator complexes appear to have a negative effect
on activated transcription, dMediator does not
exhibit such an activity in an in vitro transcription system
reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is
more plausible that the dMediator-Krüppel interaction observed
is a part of the mechanism for transcriptional activation rather than
transcriptional repression. Taken together with the fact that dMediator
is dispensable for basal transcription, the lack of defect of the
dMediator-depleted nuclear extracts on transcriptional repression by
Even-skipped protein suggests that dMediator is required mainly for the
mediation of transcriptional activation signals to the basal
transcription machinery. Very recently, developmental roles of certain
dMediator proteins found in the Drosophila genome database
have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).
Like yeast Mediator, dMediator bind with the CTD repeats of
Drosophila Pol II. This implies that though
dMediator was purified separately from Pol II, these two complexes
indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS.
Such interactions may be involved in the regulation of Pol II
preinitiation complex assembly. Related with this idea, it has been
reported that in yeast, recruitment of general transcription factors
such as TBP, TFIIB, and TFIIH to active promoters requires the function
of Mediator. Also, TFIIE interacts with the
Mediator protein Gal11. Further analyses will be
required to clarify whether these interactions, observed both in yeast
and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).
dMediator contains the protein kinase component Cdk8, which can
phosphorylate serine residues in the CTD. This catalytic kinase subunit
seems responsible, at least in part, for the Pol II phosphorylation by
dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II
repeats, suggesting the presence of a functional interaction between
these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH
has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).
Transcriptional elongation regulators NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF (FlyBase term: Spt5) from a Drosophila nuclear extract reduces the level of polymerase that pauses in the promoter proximal region of hsp70. Depletion of one Negative elongation factor E (NELF) subunit in salivary glands using RNA interference also reduces the level of paused polymerase. In vivo protein-DNA cross-linking shows that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborates the cross-linking result and shows that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF are strongly recruited to chromosomal puffs harboring the hsp70 genes. It is proposed that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation (Wu, 2003).
It is proposed that promoter proximal pausing occurs when the nascent transcript emerges from the RNA exit channel of the Pol II and is grabbed by the NELF-E subunit. Tethering of the NELF-E to the elongation complex would generate a rigid body that could restrict the movement of the Pol IIa. This model is supported by several observations. The paused polymerase is in the Pol IIa state, and NELF and DSIF only inhibit elongation by Pol IIa. In vitro transcription analysis indicates that the elongation complex is not receptive to inhibition by NELF and DSIF until the nascent transcript is ~30 nucleotides long. This length coincides approximately to the distance polymerase elongates on hsp70 before it pauses. In vitro transcription analyses indicate that DSIF and NELF associate with polymerase shortly after initiation but probably before the polymerase reaches the region of pausing. Finally, NELF-E has an RNA-binding motif that is essential for its inhibitory action (Wu, 2003 and references therein).
Although NELF and DSIF are sufficient to slow the elongation rate of purified Pol IIa, it is suspected that additional proteins are involved in stably pausing Pol II on the hsp70 promoter. In cell-free transcription reactions done with other promoters, the pausing caused by DSIF and NELF appears to be transient -- the polymerase eventually moves forward if given enough time. In contrast, several observations indicate that the Pol II on hsp70 is stably paused. The paused Pol II remains associated with the hsp70 promoter when nuclei are isolated from uninduced cells, and sarkosyl or high salt must accompany addition of nucleotides to cause the Pol II to resume elongation. In a cell-free system, Pol II remains stably paused on the hsp70 promoter for at least 30 min. GAGA factor might be involved in stabilizing the pause because mutations in the GAGA element result in a loss of paused Pol II (Wu, 2003).
Heat shock rapidly induces transcription as a result of the association of HSF with sites located upstream from the TATA element. The data suggest that HSF may activate transcription in part by causing NELF to dissociate from the Pol II. How HSF might cause the release of NELF is unclear. Phosphorylation of Pol IIa is likely to be an important step because the Pol II found in the body of the gene during heat shock is hyperphosphorylated. Phosphorylation of DSIF is another possibility as this has been observed to occur early in elongation in vitro. It is also unclear which kinase might be responsible for phosphorylating the Pol II. P-TEFb is a candidate because it associates with the hsp70 gene during heat shock induction, and HSF can be bypassed by directing a Gal4/P-TEFb fusion protein to the hsp70 promoter. No interaction, however, has been detected between P-TEFb and HSF. Recent results show that HSF associates with the mediator. Drosophila mediator contains a kinase that phosphorylates the CTD, and phosphorylation can occur synergistically with the TFIIH kinase. Perhaps HSF recruits the mediator and in turn the mediator releases the paused polymerase by phosphorylating the CTD (Wu, 2003).
The strong immunofluorescence staining observed for DSIF at heat shock loci during heat shock indicates that DSIF is associated with many of the polymerase molecules transcribing the gene. RNA polymerase initiates at a rate of once every few seconds during heat shock resulting in a train of elongation complexes traversing the gene. In the absence of NELF, DSIF might act as a positive elongation factor. Shortly after DSIF was discovered, another investigation identified DSIF as a cofactor required for reconstituting tat-dependent transcription. In this situation, DSIF appears to be stimulating elongation. DSIF has been found in a complex with another positive elongation factor called Tat-SF1. Tat-SF1 was first identified as a stimulatory factor for Tat, but subsequent results indicate that Tat-SF1 may promote elongation on cellular genes. In yeast, DSIF appears to act as either a positive or negative regulator of elongation depending on circumstances. A hypothesis that unites the positive and negative activities of DSIF considers this factor an adaptor that connects other modulators to the elongation complex. In this regard, DSIF has been shown to bind on its own to Pol II, whereas the stable association of NELF with Pol II requires the presence of DSIF (Wu, 2003 and references therein).
NELF and DSIF appear to associate with several hundred interbands in polytene chromosomes. Each interband could contain many genes. The weak staining of interbands by Hoecsht suggests that the DNA in the interbands is in a decondensed state. Residing in these decondensed regions could be genes whose primary control mechanism does not involve a disruption of chromatin structure or even assembly of the initiation complex. Instead, alleviating repression by NELF and DSIF could underlie the mechanism of activation (Wu, 2003).
The uninduced Drosophila hsp70 gene is poised for rapid activation. The rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 were examined in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms (Boehm, 2003).
Pol II is highly regulated both at the level of recruitment to promoters and in its progress through the stages of the transcription cycle. This regulation is executed through numerous associations with other proteins as Pol II enters the promoter, melts DNA, initiates transcription, begins early elongation, and eventually matures into a productive elongation complex. Pol II undergoes additional modifications, most notably phosphorylation of the CTD of its largest subunit as it progresses from its hypophosphorylated promoter entry form to the elongation phase, where it is highly phosphorylated at residues Ser2 and Ser5. These changes in phosphorylation are proposed to influence protein association, affecting not only Pol II's elongation properties but also its association with a variety of protein complexes that process pre-mRNA. Moreover, the pattern of phosphorylation is not stagnant during the elongation phases of the cycle and may be signaling specific associations. To define mechanisms involved in these processes in vivo, the rapidly and robustly activated hsp70 gene has been employed as a model (Boehm, 2003).
Technological advances of DNA-protein cross-linking and highly quantitative large-scale PCR assays were used to explore hsp70 activation kinetics of recruiting HSF, the critical Pol II kinase P-TEFb, and Pol II in vivo. The changes in Pol II were examined during the first and subsequent cycles of transcription that are triggered in response to an instantaneous heat shock of Drosophila cells. HSF recruitment occurs very rapidly, detectable at the earliest assay point of 5 s of heat shock, and reaches saturation within 75 s; this result is consistent with the rapid transcriptional response of heat shock genes and with previous, lower resolution assays of HSF recruitment. The recruitment of additional hypophosphorylated Pol II to the promoter occurs with rapidity similar to that of HSF recruitment but before an increase in Pol II phosphorylation at the promoter, which occurs by 75 s. All forms of Pol II achieve a maximal level on the promoter and gene by 5 min. The progress of Pol II across the gene can be observed, and its progress fits the known rate of elongation on Drosophila hsp70, 1.2 kb/min. Interestingly, total Pol II levels remain greater at the transcription start site than at the ORF, even during active transcription, consistent with the observation that promoter escape remains rate-limiting even during heat shock. P-TEFb, a major Pol II kinase, moves across the gene at a rate similar to that of Pol II during the first burst of transcription and thereafter remains distributed over the promoter and ORF during the full 20-min time course examined. This distribution supports a model where P-TEFb contributes to Pol II phosphorylation not only at the promoter but also during most or all of the elongation phase of transcription (Boehm, 2003).
The detection of Ser5 phosphorylation on a promoter-paused Pol II prior to gene induction corroborates a model where this phosphorylation is an early event involved in the transition from initiation of transcription to early phases of Pol II elongation. The mRNA associated with the paused Pol II molecule has previously been shown to be efficiently capped when long enough to allow access of the capping machinery. Since Ser5-P has been reported to enhance Pol II association with mRNA capping machinery and capping activity, the Ser5-P detected on paused Pol II might help to explain the efficient capping of paused RNAs. It is important to note that earlier analyses of the hsp70 gene, which determined that the paused Pol II CTD is hypophosphorylated prior to heat shock, were performed with antibodies different from those used in the present study. Importantly, the antibody generated to detect Pol IIo in those studies was directed against a peptide phosphorylated in vitro by CTK1, a yeast kinase thought to phosphorylate the CTD at serine 2. Thus, the previous analysis did not probe for the Ser5 phosphorylation reported here (Boehm, 2003).
While P-TEFb phosphorylates the CTD primarily at Ser2, it has also been shown to recognize Ser5 as a substrate. Present results suggest that the Ser5 detected in the uninduced state on hsp70 is not a result of P-TEFb activity, since P-TEFb is not detected prior to gene activation (as seen in this study). Ser5 is likely to be the substrate of the cdk7 component of TFIIH early in transcription. Indeed, cdk7 has been found in in vitro studies to be released earlier in the transcription cycle than P-TEFb. In vivo, Cdk7 is required very early in the transcription cycle and contributes to the generation of the paused Pol II on the promoter-proximal region of hsp70 (Boehm, 2003).
Analyses of the phosphorylation status of the CTD in other organisms have found Ser5-P levels to be higher at the promoter than at the ORF, a pattern similar to what was observed on hsp70 during active transcription. When total levels of Pol II are taken into account on hsp70, however, it appears that the level of Ser5-P remains constant along the gene. Comparatively, another study did not see a striking difference in total Pol II density along the genes analyzed. A third study detected more Ser5-P at the promoter than the 3' untranslated region of the human alpha-AT gene but also appeared to detect more total Pol II at the promoter region. Thus, it may be that in metazoans (or on some genes) the level of Ser5-P relative to Pol II is fairly constant along the gene. The possibility that the activity of a phosphatase may be system or gene specific is certainly plausible; for instance, heat shock of HeLa cells deactivates a CTD phosphatase (Boehm, 2003 and references therein).
Under non-heat shock conditions, total Pol II levels were greater at the 5' regions than at the ORFs for several genes that contain promoter-paused Pol II, while histone H1, which does not display a pause by nuclear run-on assay, shows no significant difference of 5' and ORF Pol II signals. Greater levels of Ser5-P were also detected at the 5' end of the genes containing paused Pol II, while levels on H1 were distributed evenly, indicating that this phosphorylation may be a general aspect of the regulatory status of a paused Pol II. This distribution of Ser5-P for the constitutively active genes Tub, GAP, and Actin5C is similar to the results of other studies which analyzed active transcription; however, Ser5-P levels on these genes are constant when standardized to total Pol II, similar to hsp70 in its active state. For these Drosophila genes, the higher level of Ser5-P at the promoter may be attributable to the presence and status of the paused Pol II, indicative of genes regulated at the level of elongation. Indeed, recent studies describe another constitutively active pause gene in human cells, dihydrofolate reductase, which shows a pattern similar to that of Ser5-P for these Drosophila genes (Boehm, 2003 and references therein).
Phosphorylation at Ser2 of the Pol II CTD may be important for processivity into active elongation and has been implicated in downstream events, including pre-mRNA splicing and 3' mRNA processing. Ser2-P levels are undetectable at +58 on hsp70 in the uninduced state, increase quickly at the 5' region upon heat shock, and appear constant through the gene in later time points (for example, 5 min). The increase in phosphorylation detected over time tracks with the recruitment of additional Pol II as well as the recruitment of P-TEFb. Taking into account total Pol II levels, there appears to be a slight increase in Ser2-P as Pol II progresses through the ORF. This correlates with the concomitant and approximately equivalent decrease in Pol IIa. Ser2-P patterns on additional genes containing a paused Pol II, when considered relative to levels of detectable total Pol II, are significantly higher in the ORF than are those in the 5' region. While these ratios may simply be a consequence of promoter-paused Pol II not being Ser2 phosphorylated, this result is similar to that of another study, where Ser2-P was only detected in the ORF. These observations led to speculation that an increase in Ser2-P may be important for cueing specific processes as Pol II proceeds through the gene. P-TEFb, the major kinase implicated in Ser2 phosphorylation, was detected concomitant with Pol II during active transcription on hsp70. While Pol II/P-TEFb ratios appear constant, a slight increase in Ser2-P occurs at the 3' end of the gene. As the presence of the kinase is not an indicator of its activity, work presently ongoing in the laboratory on P-TEFb kinase inactivation and hsp70 gene regulation should help to better understand this process (Boehm, 2003 and references therein).
Lastly, analysis of immunostaining of polytene chromosomes provides independent corroboration of the higher resolution and quantitative ChIP assays and provides insight into the formation and composition of the transcription puff. Paused Pol II on hsp70 was previously detected with this method, as was Pol II along hsp70 during heat shock. A modest detection of Ser5-P was observed on the promoter prior to heat shock. During the early stages of puff formation, Pol II resolves from promoter-bound HSF. Ser2-P and Ser5-P occupy the most decondensed regions of the puff forming a halo around the heat shock loci, while HSF is more concentrated at the chromosome core at one end of the puff. Taken together, these ChIP and immunofluorescence results provide a foundation for additional temporal and spatial assignments of specific factors relative to the phosphorylation events during the activation of transcription. Perturbation of the function or activity of specific factors using genetic and drug-based approaches will provide further insight into the mechanistic role of these factors in the recruitment and modification of transcriptional machinery and in the coupling of specific transcription processes and Pol II modifications to RNA processing events (Boehm, 2003).
Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. This study reports that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, this study has visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. Heat shock factor (HSF), the transcriptional activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. The results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. It is concluded that transcriptional activators display diverse dynamic behaviours in their associations with targeted loci in living cells. This method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo (Yao, 2006).
The rapid recovery pattern of HSF under non-heat shock (NHS) and slow recovery under heat shock (HS) corresponds in vivo to the marked difference in the DNA-binding affinity of HSF monomers (NHS) and trimers (HS). It is therefore proposed that a transcription activator's exchange dynamics on its targets may simply reflect the dissociation rate constant of the protein–promoter complex. The low affinity of some activators leads to their transient binding and has been suggested to cause the probabilistic assembly of transcriptional machinery. The high affinity of other activators leads to their stable binding, and this in turn is conducive to the formation of stable coactivator assemblies and the efficient recruitment of Pol II for repeated cycles of transcription. The exchange dynamics of some activators may involve other mechanisms; for instance, NF-kappaB, which has high affinity for DNA, was found to exchange rapidly at the tandem-repeat target gene loci. In addition, chromatin remodelling might have a function in these processes (Yao, 2006).
The slow exchange of activated HSF at the hsp70 promoter presents a sharp contrast with the rapid recruitment and elongation of RNA polymerase II at hsp70 genes during HS. During a 2-min transcription cycle (that is, the time it takes a Pol II molecule to transcribe the hsp70 gene, more than 20 Pol II molecules have begun the transcription of each hsp70 gene; however, very little new HSF has bound to the gene as shown by FRAP. Therefore, the data do not support the 'activation by destruction' hypothesis that the recruitment of new polymerase requires the ubiquitin–proteasome system (UPS) to turn over the 'spent' activator on the promoter. Moreover, more than the total amount of intracellular HSF would be degraded during a short period of heat shock if 'activation by destruction' were true for every round of heat shock gene transcription. HSF is an acidic, strong activator, like many positive regulatory factors, and hsp70 transcription resembles that of many other genes.
Recent results on the yeast Gal4 activator have shown that it, too, is stably bound to its regulatory sites during gene activation. Therefore two independent and complementary approaches on the two widely studied acidic activators have revealed their stable binding to DNA during gene activation. Alternative models for activator function that propose activator recycling as a key component, such as hit and run, chaperone-assisted disassembly or UPS-mediated turnover, can apply to some but clearly not all transcriptional activators (Yao, 2006).
The stable binding of HS-activated HSF and the transient binding of ligand-activated GR collectively show the diverse 'action modes' of transcription activators: both stably bound and transiently bound activators can support gene transcription. How individual activators function in these two modes on their respective gene targets remains to be seen, with the underlying mechanisms yet to be determined. Importantly, the dynamic behaviour of coactivators, Pol II transcription and RNA-processing machinery at native mRNA genes is largely unknown in living cells, and the described experimental approach will be applicable to further investigations (Yao, 2006).
Heat shock transcription factors (HSFs) play important roles in the cellular
response to physiological stress signals. To examine the
control of HSF activity, a yeast two-hybrid screen was undertaken for proteins interacting with Drosophila HSF. DROJ1, the fly
counterpart of the human heat shock protein HSP40/HDJ1, was identified as the dominant interacting protein (15 independent isolates
from 58 candidates). The domain of HSF interacting in the two-hybrid screen has been mapped to the region between the N- and C-terminal leucine
zippers, previously shown to modulate HSF activity. The corresponding interacting domain of DROJ1 maps to the C-terminal half of the protein and is involved in substrate binding. Overexpression of DROJ1 in Drosophila SL2 cells delays the onset of the heat shock response. Moreover, RNA
interference involving transfection of SL2 cells with double-stranded droj1 RNA depletes the endogenous level of DROJ1 protein,
leading to constitutive activation of endogenous heat shock genes. The induction level, modest when DROJ1 alone is depleted, reaches
maximal levels when DROJ1 and HSP70/HSC70, or DROJ1 and HSP90, are depleted concurrently. Chaperone co-depletion is also correlated with strong
induction of the DNA binding activity of HSF. These findings support a model in which synergistic interactions between DROJ1 and the HSP70/HSC70 and HSP90
chaperones modulate HSF activity by feedback repression (Marchler, 2001).
Continued Heat shock factor: Regulation part 2/2
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D. The Interactive Fly resides on the
Heat shock factor: Biological Overview | Evolutionary Homologs
| Effect of mutation | References
Society for Developmental Biology's Web server.