All organisms respond to cellular stress by shutting off the regular protein synthesis and

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2 1. INTRODUCTION All organisms respond to cellular stress by shutting off the regular protein synthesis and starting the synthesis of a new set of proteins called heat shock proteins or more generally, stress proteins, which help the organism cope with the stress. This induction of stress proteins is mainly at the level of transcription and the involvement of a specific heat shock transcription factor (HSF) in this induction is well documented. HSFs act by binding well conserved sequences called heat shock elements (HSEs) present in heat shock promoters. HSEs are contiguous arrays of variable number of 5bp sequence ngaan arranged in alternating orientation, where n denotes a less conserved nucleotide. At least two ngaan units are needed for recognisable binding of HSF in vitro while three repeats are bound with highest affinity, showing the cooperative nature of binding. HSFs have been cloned and characterised in several organisms. In the unstressed cell, HSF is maintained in a monomeric non DNA binding form, in both the cytoplasm and the nucleus. It is kept repressed in this form by hsp 70 which has been shown to bind HSF under non-stress conditions. Upon stress, hsp 70 mediated repression is removed and HSF homotrimer gets localised into the nucleus, binds HSEs in the heat shock gene promoters and leads to their transcriptional activation. Transcriptional activation of the heat shock genes leads to increased levels of hsp 70 and to formation again, of an hsp 70 - HSF complex, which brings back HSF to its non DNA binding monomeric form. In Drosophila melanogaster, it is known that the binding of HSFs to HSEs is greatly facilitated by another nuclear factor, which binds repeats of dinucleotide GA or TC. This factor, termed GAGA factor, binds several house keeping gene promoters and keeps them free of nucleosomes, ready for transcriptional activation, in an ATP dependant manner. All heat shock promoters in Drosophila have these GA or TC repeats in addition to the HSEs. Vl

3 2. Cloning and characterization of rat heat shock transcription factor 1 Though there are many HSFs cloned and characterized in detail, there are many gaps in our knowledge about the regulation of stress response. For instance, we do not know if there are only two HSFs in mammals. Is there a mammalian homolog of chicken HSF3, and if it is present, what its function is, is an interesting question to ask. In addition, information about the structure and expression of homologs of HSF1 from mammals other than mouse and human beings will be useful in understanding the stress response. Towards answering these questions, a rat liver edna library was screened using the radiolabeled mmhsf1. as a probe, assuming that the homology found between the mmhsf1 and HsHSF1 extends to rat HSF1 also, at least in the well conserved DNA binding domain. Using the approach mentioned above, a 1.65kb long fragment of the heat shock transcription factor 1 (HSF1) from a rat liver edna library was cloned. Using convenient restriction sites, it was subcloned and sequenced using Sanger's dideoxy method. The sequence compa~ison revealed that the homology between rat HSF1 and mmhsf1 is 96% while it is 82% between rat HSF1 and HsHSF1. There is a patch of 66 nucleotides in the rhsf1 which is missing in the mmhsf1, between the C terminal leucine zipper and the transcriptional activation domain. The presence of the trinucleotide CAG at the 3' end of the additional stretch of rhsf1 indicates that this additional stretch could arise from alternative splicing. A model for alternative splicing in HSFs has been proposed based on these results. In addition to these changes, there is a stretch of 15 nucleotides in HsHSF1 which is missing in both rat HSF1 and mmhsf1. Apart from these major changes, only six aminoacids are variant in the rat HSF1 as compared to the mmhsf aminoacid sequence. In order to study the expression of rat HSF1 in different tissues of an adult rat, an RNA dot blot hybridization analysis was done. The results of dot blot analysis revealed that rhsf1 is expressed at a slightly higher level in the heart, followed by brain, ovary, spleen, etc. Vll

4 3. Identification and characterization of the promoter elements responsible for the heat inducibility of the rat albumin promoter The expression of heat shock proteins is not limited as a response to stress. Many hsps are known to be expressed in the normal course of development, in a tissue and time specific manner. Srinivas eta/ (1987) had reported that heat shock given to the early embryonic rat liver results in an increase in the amounts of albumin at a premature stage. HSF independent activation of stress genes also is known, although in very few cases, in S. cereviseae. Both cytosolic catalase T (CTT1) and DDR2 genes in yeast are known to be heat inducible but their promoters lack HSEs. Now it is known that they both employ different cis and trans elements for this heat induction. It was thus interesting to find out if the induction of albumin gene upon heat shock in the early stages of rat liver development follows the classical, HSF mediated pathway or not. The second part of the present study addresses this question. In order to study the heat inducibility of rat albumin promoter in the early embryonic stages of development, a cultured cell model system similar to embryonic rat liver cells was needed. H411E-C3 cell line is a rat hepatoma cell line in which the rate of transcription of albumin and other liver specific genes is about 5-10% of the adult liver cells. This low rate of 5-10% corresponds well with the rate of transcription of albumin gene in normally developing embryonic liver cells at days of gestation. Hence, it was thought that 6 H411E-C3 cells may serve as a useful model system for our purpose. This choice of cell line was confirmed using western blots which show that the level of albumin goes up in H411E-C3 cells upon heat shock at 44 C for 45min. Vlll

5 In order to quantify this increase in the amount of albumin in H411E-C3 cells upon heat shock and also to determine whether the incresase seen in the amounts of albumin upon heat shock in H411E-C3 cells is a result of transcriptional regulation or post-transcriptional regulation in response to stress, transient transfection assays followed by CAT assays were designed, with expression vectors carrying the bacterial CAT gene under the influence of different lengths of rat albumin promoter. An expression vector in which the CAT gene is under the control of a 390bp rat albumin promoter fragment was available in the laboratory (Aib390-CAT, a kind gift of Dr. Moshe Yaniv). Since heat shock elements are capable of influencing the rate of transcription from long distances, it was worthwhile studying longer fragments of rat albumin promoter. Towards this, a longer fragment (440bp) of the rat albumin promoter was obtained using PCR. Using this albumin promoter fragment, expression vectors in which the 440bp fragment of albumin promoter is placed upstream of a CAT gene in either right orientation (Aib440-CAT1 and Alb440-CAT5) or a reverse orientation (Aib440-CAT2) were constructed. These constructs were used in transient transfection assays through electroporation, following which cells were given appropriate heat shock and CAT assays were done with the extracts prepared from these cells. The CAT assay results show that there is an increase in CAT activity in the cells transfected with both Alb390-CAT and Alb440-CAT1, upon heat shock at 44 C for 45 min. However, the increase in CAT activity seen upon heat shock in Alb440-CAT1 transfected cells was higher than the increase seen with Alb390-CAT. There was no CAT activity in Alb440-CAT2 (where albumin promoter is in the reverse orientation) transfected cells as expected, either before or after heat shock, where as positive control hsp70-cat construct transfected cells showed an increase in CAT activity upon heat shock. These results suggest that there is a functional HSE within the -390bp region in addition to the one at around -440bp and that both contribute to the elevated expression of albumin seen upon heat shock in the early embryonic rat liver cells. lx

6 In order to understand the mechanism of early induction of albumin in developing embryonic rat liver cells, the rat albumin promoter sequence was analysed for the presence of HSEs or HSE like sequences and a few HSE like sequences were found within -500 bp region. These HSEs were found to be similar in number, position and extent of conservation, to those in known hsp promoters like Drosophila hsp83 and yeast hsp26. One element at -440 region was more conserved and it was decided to further study this region for its ability to interact with heat shock transcription factors. Gel retardation assays were then done with H411E-C3 or adult liver cell extracts and radiolabeled HSEs in the rat albumin promoter, which showed that the HSEs in the -440bp position of rat albumin promoter bind the HSFs, both from the liver cells and in the H411E-C3 cells. That this interaction is sequence specific and reversible in nature was proved by competition experiments where 100 molar excess of non-labeled HSEs were included in the gel retadardation reactions. In addition to HSEs, repeats of dinucleotides GA or TC are characteristic features of all heat shock promoters. These repeats are recognised by the GAGA factor which disrupts the nucleosomes and keeps the promoter ready for HSF occupancy and transcriptional activation. Though some mammalian GAGA factors are reported, their exact function is not known in mammals. It was thus interesting to analyze the rat albumin promoter for the presence of GA or TC repeats. It was found that at around -250bp position, there are seven such contiguous repeats, which is a potential GAGA factor binding site. Gel retardation assays done with either liver cell extracts or the H411E-C3 cell extracts and radiolabeled GAGA repeats from the rat albumin promoter showed the formation of two complexes in a sequence specific and reversible manner. One of these two complexes was found both in the normal cells as well as the heat shocked cells while the other complex was specific to normal cells. It is not yet clear if these two complexes correspond to two X

7 different forms of the same factor or they are two different factors altogether. However, these results indicate that the rat albumin promoter contains the GAGA repeats which are recognised by specific nuclear factors. The role of these factors is yet to be ascertained. In an adult rat, expression of albumin is limited to liver. In other tissues, dominant negative factors are thought to suppress the albumin gene transcription. In order to find out if heat shock activated HSF can override this suppression of albumin gene in other tissues of an adult rat, different tissue slices from an adult rat were subjected to heat shock, total RNA was isolated from these cells and a northern blot was done using radiolabeled albumin cdnaas probe. The results show that HSF fails to overcome the suppression mediated by the dominant negative suppressors of albumin gene transcription in adult tissues other than liver. Thus, the heat mediated activation of albumin gene transcription in developing liver cells is in coordination with other tissue specific factors and not independent of them. 4. CONCLUSIONS: 1. Rat HSF1 sequence is highly homologous to the mouse HSF1 sequence, but for the presence of a stretch of 66 nucleotides in rhsf1 which is absent in mmhsf1. Aternative splicing occurring in HSFs is a possible source of this additional stretch. 2. Rat HSF1 is expressed at a slightly higher level in the heart, followed by brain, ovary, spleen etc. 3. Rat albumin promoter activity is modulated by heat shock in the early stages of embryonic development of liver. 4. There are two HSEs in the rat albumin promoter - one at -440bp position and the other within -390bp position. HSFs bind the HSE at -440bp in a sequence specific and reversible manner. 5. There is a GAGA factor binding site at around -250bp position in the rat albumin promoter which is bound by two specific nuclear factors in a sequence specific and revrsible manner. 6. Heat shock fails to overcome the negative repression of albumin gene transcription in different tissues other than liver in an adult rat.. Xl