MECHANISM OF HEAT INDUCTION OF ALBUMIN IN EARLY EMBRYONIC RAT-LIVER

Albumin is an adult liver specific protein whose induction in rats sta rts at day 19 or 20 of normal gestation. Our studies on the effect of heat stress on embryonic development showed premature induction of a 6 7 kDa protein at day 12 or 13 in embryonic liver cells, in addition to the induction of usual heat shock proteins. Immunoblotting with anti- albumin antibody confirmed the prematurely induced protein to be album in. RNA dot blot showed that albumin induction upon heat shock is regu lated at transcriptional level and northern blot confirmed the size of heat induced albumin transcript to be similar to the constitutively i nduced albumin RNA transcript. During heat stress, heat shock proteins are induced by the interaction of a specific heat shock transcription factor (HSF) with specific DNA sequences (heat shock elements, HSEs) present in the promoters of all heat shock genes. The functional signi ficance of HSF-HSE interaction is confirmed by transient transfection assays using plasmids carrying chloramphenicol acetyl transferase repo rter gene under the control of different deletion fragments of the rat albumin promoter. These assays identified the HSEs to be within -450 base pairs of the rat albumin promoter. Deletion of these HSE sequence s from rat albumin promoter abolished its heat inducibility. Electroph oretic mobility shift assays with synthetic oligonucleotides, represen ting putative HSEs in the rat albumin promoter, and H4II-E-C3 cell ext racts showed that the heat shock factor binds this region in a sequenc e specific and reversible manner. Gel super-shift assays with antibodi es to HSF1 and HSF2 demonstrated that the HSEs present in the rat albu min promoter are bound by HSF1 but not by HSF2. In addition to the HSE s, we have identified a putative GAGA factor binding site in the rat a lbumin promoter at -228 bp to -252 bp position. These GAGA repeats are bound in a sequence-specific and reversible manner by two factors in non-stressed cells, whereas only one of these two factors continues to bind the GAGA repeats under heat shock conditions. We thus show that rat albumin promoter contains (i) functional HSEs to which the HSF1 bi nds and (ii) GAGA factor binding sites to which the GAGA factor binds and that the promoter activity can be modulated by temperature.