MCAT Practice Test 5

Heat-induced expression of a chaperone gene

¹When human cells are exposed to elevated temperature, misfolded proteins accumulate and the cells increase synthesis of molecular chaperones. Researchers studying a cultured human epithelial cell line proposed that the transcription factor TRF4 (55 kDa) is required for heat-induced expression of CHP7, a 70-kDa chaperone. TRF4 binds DNA as a homodimer at a promoter sequence called the thermal response element (TRE), which is present upstream of the CHP7 gene.

²Cells were transfected with either a nontargeting control siRNA or an siRNA complementary to TRF4 mRNA. After 48 h, cells were kept at 37 °C or shifted to 42 °C for 2 h. CHP7 mRNA was measured by reverse transcription–quantitative PCR (RT-qPCR) and expressed relative to the 37 °C control-siRNA sample (Figure 1). Protein levels in the same four conditions were analyzed by Western blot with antibodies against TRF4, CHP7 and β-actin (Figure 2).

³In a separate experiment, chromatin immunoprecipitation (ChIP) with an anti-TRF4 antibody showed that the amount of TRE DNA bound by TRF4 increased about sixfold within 30 min of the shift to 42 °C in cells given control siRNA. Immunofluorescence in the same cells showed that TRF4, which is mostly cytoplasmic at 37 °C, accumulates in the nucleus after the temperature shift.

⁴The researchers also characterized R212W, a TRF4 missense allele found in one family. In vitro, the R212W protein dimerizes normally with wild-type TRF4 but cannot bind the TRE, and any dimer that contains at least one R212W subunit fails to activate transcription. Family members heterozygous for the allele showed a reduced CHP7 response to fever.