Genome-Wide Role of HSF1 in Transcriptional Regulation of Desiccation Tolerance in the Anhydrobiotic Cell Line, Pv11

Abstract

The Pv11, an insect cell line established from the midge Polypedilum vanderplanki, is capable of extreme hypometabolic desiccation tolerance, so-called anhydrobiosis. We previously discovered that heat shock factor 1 (HSF1) contributes to the acquisition of desiccation tolerance by Pv11 cells, but the mechanistic details have yet to be elucidated. Here, by analyzing the gene expression profiles of newly established HSF1-knockout and -rescue cell lines, we show that HSF1 has a genome-wide effect on gene regulation in Pv11. The HSF1-knockout cells exhibit a reduced desiccation survival rate, but this is completely restored in HSF1-rescue cells. By comparing mRNA profiles of the two cell lines, we reveal that HSF1 induces anhydrobiosis-related genes, especially genes encoding late embryogenesis abundant proteins and thioredoxins, but represses a group of genes involved in basal cellular processes, thus promoting an extreme hypometabolism state in the cell. In addition, HSF1 binding motifs are enriched in the promoters of anhydrobiosis-related genes and we demonstrate binding of HSF1 to these promoters by ChIP-qPCR. Thus, HSF1 directly regulates the transcription of anhydrobiosis-related genes and consequently plays a pivotal role in the induction of anhydrobiotic ability in Pv11 cells.

Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11

“…In contrast, the AcGFP1 expression unit was detected only on chromosome 1 (Chr1; Figure 2 B,C, and Data S9 and S10 ) in both clones. Three of these integration sites, Chr1:280397, Chr1:21155382 and Chr1:21164645, were located in intergenic regions, while a fourth, Chr1:21143572, was located in the intron of transcription unit, g12121 , whose expression is relatively low in Pv11 cells ( Figure 2 C, and Table S7 , accession number GSE171333 [ 17 ]).…”

“…Next, we examined whether genomic integration at the above sites affects the anhydrobiotic ability or proliferation rate of the corresponding cells. As shown in Figure 3 A, AcGFP1 and ZeoR expression units were inserted individually into each genomic site in wild-type Pv11 cells by the CRIS-PITCh method [ 17 , 18 , 22 ]. Although precise integration was not achieved at Chr1:280397 ( Figure S2 ), the other three sites allowed exogenous DNA integration as designed, and this was confirmed by Sanger sequencing ( Data S11–S16 ).…”

“…A donor vector containing bidirectional HaloTag and AcGFP1 expression unit was designed, the former as an example of a GOI, and the latter as a marker of successful integration ( Figure 5 A). In our first attempt, a 40 bp homology arm length was used as shown in Figure 3 and described in previous studies [ 17 , 18 , 22 ]. However, the integration efficiency was low (7.1 ± 1.3% of the target cells were HaloTag + /AcGFP1 + cells; Figure 5 B,C), possibly because the insert size was much longer than the homology arm length.…”

“…For Cas9 expression, the previously constructed vector, pPv121-hSpCas9, was used; gRNA expression vectors were also constructed as reported previously [ 17 ]. Briefly, pPvU6b-DmtRNA-BbsI was digested with BbsI, and annealed oligonucleotides were ligated into the cut vector.…”

“…To explore the molecular mechanisms underlying the desiccation tolerance of Pv11 cells, we have exploited several gene-manipulation techniques, including a random integration method [ 16 ] and a site-specific knock-in method using the CRISPR/Cas9 system [ 17 , 18 ], and have generated several key resources. We have produced a transgenic cell pool, so-called KH cells, by random integration of an AcGFP1 ( Aequorea coerulescens GFP)-expressing plasmid [ 16 ].…”