Heat shock factor 1 is inactivated by amino acid deprivation

Abstract

Mammalian cells respond to a lack of amino acids by activating a transcriptional program with the transcription factor ATF4 as one of the main actors. When cells are faced with cytoplasmic proteotoxic stress, a quite different transcriptional response is mounted, the heat shock response, which is mediated by HSF1. Here, we show that amino acid deprivation results in the inactivation of HSF1. In amino acid deprived cells, active HSF1 loses its DNA binding activity as demonstrated by EMSA and ChIP. A sharp decrease in the transcript level of HSF1 target genes such as HSPA1A (Hsp70), DNAJB1 (Hsp40), and HSP90AA1 is also seen. HSPA1A mRNA, but not DNAJB1 mRNA, was also destabilized. In cells cultured with limiting leucine, HSF1 activity also declined. Lack of amino acids thus could lead to a lower chaperoning capacity and cellular frailty. We show that the nutrient sensing response unit of the ASNS gene contains an HSF1 binding site, but we could not detect binding of HSF1 to this site in vivo. Expression of either an HSF1 mutant lacking the activation domain (HSF379) or an HSF1 mutant unable to bind DNA (K80Q) had only a minor effect on the transcript levels of amino acid deprivation responsive genes.

Fig. 1

HSF1 loses its DNA binding affinity upon leucine starvation. a QPCR validation of ASNS, DNAJB1, and HSPA1A mRNA levels relative to GAPDH mRNA levels upon leucine starvation. Error bars represent SD; *P < 0.05; ***P < 0.001, relative to +Leu. b HEK293 cells were deprived of leucine for 24 h. Cytoplasmic (Cyt) and nuclear (Nuc) extracts were made and subjected to SDS–PAGE and western blot analysis using an anti-HSF1 antibody. c Nuclear extracts were subjected to 2D gel electrophoresis and western blot analysis using an anti-HSF1 antibody. d Nuclear extracts were used in an electrophoretic mobility shift assay with a double-stranded oligonucleotide with the HSE sequence. Supershifts were induced with an anti-HSF1 antibody. Single arrows indicate the primary complexes formed; double arrows indicate the supershifted complexes. e Chromatin immunoprecipitation was performed using an anti-HSF1 or an anti-ATF4 antibody. Bound chromatin was analyzed by QPCR using a primer set surrounding the HSE of the DNAJB1 promoter. As a control, the ChIP was performed without an antibody. Error bars represent SD; **P < 0.01, relative to +Leu

Fig. 2

HSP mRNA and HSP protein levels are decreased upon leucine starvation. a HEK293 cells were starved for leucine and harvested at the indicated time points. mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. b Nuclear extracts were used in an EMSA with a double-stranded oligonucleotide with the HSE sequence. The arrow indicates the primary complex formed. c HEK293 cells were starved for leucine and after 30 min 5 μg/ml actinomycin D was added to block transcription. Cells were harvested at the indicated time points. mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. d Lysates were subjected to SDS–PAGE and western blot analysis using antibodies against the indicated proteins

Fig. 3

Leucine starved cells can still respond to a proteotoxic insult. a HEK293 cells were deprived of leucine for 24 h and exposed to a heat shock for 30 min at 45°C. Cytoplasmic (Cyt) and nuclear (Nuc) extracts were made and subjected to SDS–PAGE and western blot analysis using an anti-HSF1 antibody to determine HSF1 localization. The results obtained with extracts from unstressed cells isolated in parallel are shown in Fig. 1b. b Nuclear extracts of stressed and unstressed cells were used in an electrophoretic mobility shift assay with a double-stranded oligonucleotide with the HSE sequence. Supershifts were induced with an anti-HSF1 antibody. Single arrows indicate the primary complexes formed; double arrows indicate the supershifted complexes

Fig. 4

Lysine and glutamine starvation also inactivate nuclear HSF1. a HEK293 cells were cultured in the presence of all amino acids (+) or deprived of lysine or glutamine (−) for 24 h. EMSA was performed with a double-stranded oligonucleotide with the HSE sequence. Supershifts were induced with an anti-HSF1 antibody. Single arrows indicate the primary complexes formed; double arrows indicate the supershifted complexes. b ASNS and HSPA1A mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. Error bars represent SD; *P < 0.05; ***P < 0.001, relative to +amino acid

Fig. 5

Amino acid limitation affects HSF1 binding affinity and HSP mRNA levels. a HEK293 cells were cultured in medium containing limiting amounts of leucine for 24 h. The concentrations are indicated relative to the standard leucine concentration in medium (+), which is 450 μM. EMSA was performed with a double-stranded oligonucleotide with the HSE sequence. The arrow indicates the primary complexes formed. b HEK293 cells were cultured for the indicated times in medium containing 45 μM leucine. As a control, cells were cultured in parallel in standard medium containing 450 μM leucine (+). Medium was changed every day. Nuclear extracts were used in EMSA with a double-stranded oligonucleotide with the HSE or NSRU sequence. The closed arrow indicates the HSE complex formed. Open arrows indicate the NSRU complexes formed. c ASNS, DNAJB1, and HSPA1A mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. Error bars represent SD; *P < 0.05; **P < 0.01; ***P < 0.001, relative to 0 days

Fig. 6

The ASNS NSRU contains a HSE. a Sequence of the NSRU of the ASNS promoter. The putative HSF1 binding sequence is underlined. b Sequence of a tandem repeat of the nutrient sensing response unit of the ASNS promoter used in the reporter plasmid. The putative HSF1 binding sequence is underlined. NSRU 1xmut and NSRU 2xmut are the sequences that are mutated for the putative HSE (indicated in bold). c HEK-dnHSF1 cells were transfected with the indicated NSRU reporter plasmid and treated with doxycycline. Cells were harvested and assayed for reporter gene activities. The results shown are the average of three independent transfections. d HEK-wtHSF1 cells were cultured in the presence of doxycyline and heat stressed for 30 min at 45°C. Directly after heat stress, nuclear extracts were made. EMSA was performed with a double-stranded oligonucleotide with the 2xNSRU sequence. Where indicated, a 2-fold molar excess of unlabeled double-stranded oligonucleotide with the 2xNSRUmut sequence was added. Supershifts were induced with an anti-HSF1 antibody. Open arrows indicate the ATF4 complexes; closed arrows indicate the HSF1 specific complex and the supershifted complex. e Doxycycline treated HEK-wtHSF1, HEK-HSF1 K80Q, and HEK-dnHSF1 cells were starved for leucine. mRNA levels were determined by QPCR analysis and are shown relative to GAPDH mRNA levels. f, g Chromatin immunoprecipitation was performed using an ATF4 antibody. Bound chromatin was analyzed by QPCR using a primer set surrounding the NSRU of the ASNS promoter. In all figures, error bars represent SD; *P < 0.05; **P < 0.01; ***P < 0.001