Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas

Abstract

SummaryWe have shown previously that the HSP70A (A) promoter, when fused upstream of other promoters, signi®cantly improves their performance in driving transgene expression in Chlamydomonas. Here, we employed the bacterial resistance gene ble, driven by the RBCS2 (R) promoter or an AR promoter fusion, to determine, by which mechanism(s) the A promoter may exert its enhancing effect. We observed that transformation rates of AR-ble constructs were signi®cantly higher than those of R-ble constructs. However, ble mRNA levels in pools of transformants generated with either construct type were the same. Co-transformation experiments revealed that the R-ble transgene was silenced in 80% of the transformants, whereas this fraction was reduced to 36% in transformants harbouring the AR-ble transgene. We conclude that the A promoter acts by decreasing the probability that a transgene becomes transcriptionally silenced. We mapped two elements within the A promoter that are responsible for this effect. The core of the ®rst element appears to be located between nucleotides ± 7 and + 67 relative to the HSP70A transcriptional start site. Its activity is strongly dependent on its spatial setting with respect to the R promoter and is increased by upstream sequences (± 196 to ± 8). The second element is independent of the ®rst and is located to the region from ± 754 to ± 197. Its activity is spacing-independent and additive to the ®rst element.

Transcription Factor–Dependent Chromatin Remodeling at Heat Shock and Copper-Responsive Promoters in Chlamydomonas reinhardtii

“…Heat shock leads to a closed chromatin structure, as levels of histone H3/4 acetylation strongly decrease and nucleosome occupancy increases. These results suggested reduced activity of the RBCS2 promoter under heat stress conditions, which indeed was observed previously for RBCS2 promoter driven transgenes that are much more weakly expressed than the endogenous RBCS2 gene (Schroda et al, 2002). Expression levels also of other Chlamydomonas genes were observed to decline during heat stress (Dorn et al, 2010).…”

“…The data on the promoter regions correspond to that shown in Figures 4 and 7. In case of ChIP analysis with antibodies against modified histones, an additional A remarkable property of the HSP70A promoter is that in a transgene setting it strongly increases the likelihood that a promoter fused downstream becomes active (Schroda et al, 2000(Schroda et al, , 2002. This effect is dependent on the presence of HSEs within the HSP70A promoter, suggesting that it is mediated by HSFs (Lodha et al, 2008).…”

A New Assay for Promoter Analysis in Chlamydomonas Reveals Roles for Heat Shock Elements and the TATA Box in HSP70A Promoter-Mediated Activation of Transgene Expression

“…We found that transgene expression in Chlamydomonas was significantly improved when the Chlamydomonas HSP70A (A) promoter was fused upstream from the transgene-driving promoter (12,13,14). The A promoter apparently acted as a transcriptional state enhancer; i.e., it improved the probability of randomly integrated transgenes becoming expressed (10,14). Enhancing activities could be assigned to two different regions of the A promoter, a proximal region (ranging from bp Ϫ23 to Ϫ285 upstream from the translational start codon) and a distal region (upstream from bp Ϫ286).…”

“…To this end, A promoter deletions and mutations were fused upstream from the RBCS2 promoter (R) to drive expression of the ble gene, conferring resistance to zeocin (9). In earlier work, we estimated the activation efficiency of A promoter derivatives (i) by counting zeocin-resistant colonies produced by cells directly transformed with R-ble/AR-ble constructs and (ii) by determining the fraction of ble-expressing, zeocin-resistant arginine-prototrophic transformants that emerged from arginine-auxotrophic cells cotransformed with the ARG7 gene and R-ble/AR-ble constructs (14). As these methods were tedious and/or led to statistically insignificant results, we sought for an alternative assay to quantify the activating effect of the A promoter.…”

Development of an expression system using the heat shock protein 70 promoter in the red macroalga, Porphyra tenera

“…HSP70 promoters, which provide both constitutive and stress-inducible expression, are widely utilized to express heterologous and homologous genes in eukaryotic organisms (Medford et al 1989;Schroda et al 2000). In the green alga, Chlamydomonas, the HSP70 promoter allows efficient transgene expression due to its constitutive expression and suppression of transgene silencing by sequence elements in the promoter (Schroda et al 2002).…”