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

Abstract

The aim of this work was to identify cis-regulatory sequences within the Chlamydomonas HSP70A promoter that mediate its stimulatory effect on the expression of downstream promoters. For this, we deleted/mutated the HSP70A promoter and, using a new assay, quantified its stimulatory effect. Our results indicate that the effect is mediated largely by heat shock elements and the TATA box.Transgenic approaches with Chlamydomonas often suffer from the low percentage of transformants that express a stably integrated transgene (e.g., 4, 5, 13). 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).The goal of this work was to identify cis-regulatory sequences within the A promoter by means of which it mediates activation of transgene expression. 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. We reasoned that it might be possible to quantify the amount of transgene transcript produced per intact transgene in pools of hundreds of cotransformants generated with the ARG7 gene and R-ble/AR-ble constructs. As in our hands around 20 to 60% of cotransformants contain the cotransformed construct and in case of R-ble ϳ20% of the transgenes are expressed (14), the R-ble construct was expected to be expressed in only 4 to 12% of the cotransformants. To test whether Northern analysis was sensitive enough to detect such low ble mRNA levels in pools of cotransformants, a liquid culture in TAP medium (6) of a Chlamydomonas strain containing an expressing AR-ble construct was increasingly diluted with a culture of a strain containing a nonexpressing R-ble construct (all transformants were generated with strain cw15-302, kindly provided by R. Matagne, University of Liège, Belgium). Northern analysis of RNA extracted from these cells (as described in reference 7) revealed that ble transcript was detectable when only 1% of the cells expres...

Function and regulation of the glutathione peroxidase homologous gene GPXH/GPX5 in Chlamydomonas reinhardtii

“…Alternatively, the loss of binding of transcription factors to the mutated Inr element could alter chromatin structure and thereby reduce the binding of other transcriptional activators to the promoter. A similar effect was suggested for a mutation of the TATA-box on HSP70A-mediated transgene activation (Lodha et al 2008). The consensus sequence of a CAAT box is CCAAT which is not 100% identical to the two elements found in GPXH, even though some heterogeneity for the first base was reported in plants (Shahmuradov et al 2003).…”

A Mediator of Singlet Oxygen Responses in Chlamydomonas reinhardtii and Arabidopsis Identified by a Luciferase-Based Genetic Screen in Algal Cells

“…The HSP70A promoter driving the luciferase reporter is not only responsive to 1 O 2 , but it is also strongly inducible by heat shock. As the heat response is genetically distinct from the response to 1 O 2 (Shao et al, 2007;Lodha et al, 2008;Shao and Bock, 2008), any mutant specifically affected in 1 O 2 responses should still show wild-type-like luciferase induction by heat. This was the case for 12 of the algal mutants identified in the MB screen ( Figure 1C), suggesting that these 12 mutants are neither generally affected in luciferase expression nor generally defective in their response to abiotic stresses.…”

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

“…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). As the constitutively high acetylation levels at the HSP70A promoter depend on HSF1, it is tempting to speculate that in the transgene setting the activation of downstream promoters is mediated by histone acetyltransferase activities recruited by HSF1.…”

“…HSEs contain at least three 59-nGAAn-39 repeats in alternating orientations and are present in the promoters of heat shock genes in a diverse set of organisms (Pelham, 1982). As deletion of HSEs from the Chlamydomonas HSP70A promoter entirely abolishes its heat shock inducibility, HSEs are also clearly indispensable for the regulation of the heat shock response in Chlamydomonas (Lodha et al, 2008). Chlamydomonas contains a single canonical HSF (HSF1), which possesses all features typical for plant (class A) HSFs and represents a key regulator of the stress response in this alga (Schulz-Raffelt et al, 2007).…”