BMC Genomics 2008, 9:298 http://www.biomedcentral.com/1471-2164/9/298 Influence of stress and growth conditions on the transcriptome The detection of a significant cohort of developmentally regulated trypanosome mRNAs by microarray, and their validation by qRT-PCR, provided confidence that our pro- cedure could be exploited for investigation of transcrip- tome changes associated with differing conditions. In several model organisms, the interactions between gene products has been probed by analysis of coordinated changes to the transcriptome in response to challenges to the cell. Such studies have included several changes to growth conditions, insult with specific chemicals to invoke the unfolded protein response (UPR), and targeted manipulations of gene expression [101-104]. Here we used all three approaches to probe the T. brucei transcrip- tome in order to determine the level of flexibility, and to assess if such data could facilitate construction of interac- tion and pathway maps based on coordinate expression profiles. Iron starvation Bloodstream trypanosomes are sensitive to iron starva- tion, and upregulate ESAG6/7 transferrin receptor (TfR) mRNA and protein levels when placed in low transferrin or iron-depleted media [45,105] and adjust TfR expres- sion to compensate for reduced endocytosis [65]. Both observations suggest a specific transcriptional-level response resulting from an iron-sensing mechanism. The transcriptomes of BSFs cultured in different proportions of fetal bovine serum (FBS), in the absence of FBS or in the absence of FBS but supplemented with bovine holo-trans- ferrin were analysed. An increase in ESAG6/7 mRNA was detected under FBS depletion by the array (Table 2), which was validated by qRT-PCR in agreement with previ- ous analyses [45,105]. mRNAs for procyclin, the gp63 iso- form TbMSP-C (Tb10.70.5290) and the major cysteine peptidase (Tb927.6.960) were upregulated in serum- depleted cells (Table 2). In cells grown in 30% FBS, procy- clin mRNA, as well as an ISG-like protein (Tb927.5.410) and a putative calcium-dependent protein kinase (Tb927.4.3770) were downregulated (Table 2), which contrasts with a moderate upregulation of procyclin in serum free conditions. These alterations were confirmed by qRT-PCR, and may suggest a suppression of procyclin expression by serum factors. However, overall there were no additional transcriptome changes when cells were grown in 30% FBS or in 0% FBS with or without transfer- rin. Scatter plots of the entire array data set indicate few transcripts that fall outside of the region considered as equivalent, in marked contrast to the scattergram obtained for developmental changes (compare Figure 4B–D with Figure 4A or Figure 1). These data suggest a very limited and specific ability of the BSF to respond to alterations in levels of nutrients, which may be restricted primarily to ESAG6/7. Unfolded protein response (UPR) Transcriptome responsiveness was further tested by attempts to invoke the UPR, a classic ER-based pathway stimulated by the presence of increased levels of unfolded polypeptides [106]. The UPR is mediated via transcrip- tional responses in both yeast and metazoan cells [104,107], and can be reliably activated by addition of tunicamycin or dithiothreitol (DTT). Using tunicamycin and DTT at concentrations that invoke a UPR in mamma- lian cells, Arabidopsis, yeast and other systems [108-112], we found that, in T. brucei, tunicamycin resulted in growth arrest over a period of up to 24 hours, while DTT exposure led to rapid cell death [see Additional file 4]. This concen- tration of tunicamycin also efficiently inhibits trypano- some N-glycosylation [113]. Therefore, we analysed the transcriptome at 5 μg/ml and 1 mM for tunicamycin and DTT, respectively, under conditions where cells remained viable, as assessed by motility. Essentially no significant change to the transcriptome was observed by either treatment. Very few ORFs showed sig- nificant differential regulation under DTT or tunicamycin treatment, and scatterplots indicate few transcripts falling outside of the equivalence region, in marked contrast to developmental changes (Table 2, Figure 4E–H). Only one hypothetical chaperone was upregulated after 4 hr in 1 mM DTT, while Rab11, ESAG7, and an ISG-like protein (Tb927.5.410) were downregulated. By contrast, a PDI class ORF, Tb10.6k15.2290, was downregulated after 24 hr of tunicamycin treatment, while ESAG6/7 as well as procyclin were upregulated in cells treated with tunicamy- cin (Table 2). Further, total cell lysates were examined for increased expression of the major ER chaperone, BiP, by Western blotting and qRT-PCR. No alteration in expres- sion of this marker, which is rapidly and strongly induced by the UPR in higher eukaryotes, was observed following DTT or tunicamycin exposure [see Additional file 4]. These data indicate an essential lack of transcriptome responsiveness to altered conditions by the trypanosome, and specifically the complete absence of a classical UPR. Significantly, this likely contributes to the extreme sensi- tivity of trypanosomes to DTT. Knockdown of clathrin and VSG As an additional strategy for investigating transcriptome responsiveness, the mRNAs of two highly important pro- teins involved in endocytosis and surface architecture, specifically the clathrin heavy chain (CLH) and VSG, were suppressed with RNAi. Both proteins are essential and CLH RNAi results in rapid cell death. For CLH, knock- down leads to a complete block to endocytosis [114], while VSG RNAi results in arrest of cell cycle progression [115]. We hypothesized that if trypanosomes were able to sense alterations in trafficking and respond to these Page 13 of 28 (page number not for citation purposes)