Change in DNase I accessibility at the promoter and upstream region of <i>OsDREB1b loci</i>.

<p>Relative DNase I accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with PCR based method. Nuclei were digested with DNase I (5 U/ml) for increasing time period (0,3,6,10 min). The isolated DNA was used for PCR reaction with primers specific for promoter and upstream region. The amount of DNA amplified at each time point was normalised to that at time 0 and plotted against time to compare the rate of degradation. The relative rate of accessibility for actin promoter (A and D) and <i>OsDREB1b</i> (B, C, D and E) and The data represented here is a mean of three independent experiments with standard error bars. Statistically significant values were marked with *.</p

Differential Acetylation of Histone H3 at the Regulatory Region of <i>OsDREB1b</i> Promoter Facilitates Chromatin Remodelling and Transcription Activation during Cold Stress

<div><p>The rice ortholog of DREB1, <i>OsDREB1b</i>, is transcriptionally induced by cold stress and over-expression of <i>OsDREB1b</i> results in increase tolerance towards high salt and freezing stress. This spatio-temporal expression of <i>OsDREB1b</i> is preceded by the change in chromatin structure at the promoter and the upstream region for gene activation. The promoter and the upstream region of <i>OsDREB1b</i> genes appear to be arranged into a nucleosome array. Nucleosome mapping of ∼700bp upstream region of <i>OsDREB1b</i> shows two positioned nucleosomes between −610 to −258 and a weakly positioned nucleosome at the core promoter and the TSS. Upon cold stress, there is a significant change in the nucleosome arrangement at the upstream region with increase in DNaseI hypersensitivity or MNase digestion in the vicinity of <i>cis</i> elements and TATA box at the core promoter. ChIP assays shows hyper-acetylation of histone H3K9 throughout the locus whereas region specific increase was observed in H3K14ac and H3K27ac. Moreover, there is an enrichment of RNA PolII occupancy at the promoter region during transcription activation. There is no significant change in the H3 occupancy in <i>OsDREB1b</i> locus negating the possibility of nucleosome loss during cold stress. Interestingly, cold induced enhanced transcript level of <i>OsDREB1b</i> as well as histone H3 acetylation at the upstream region was found to diminish when stressed plants were returned to normal temperature. The result indicates absolute necessity of changes in chromatin conformation for the transcription up-regulation of <i>OsDREB1b</i> gene in response to cold stress. The combined results show the existence of closed chromatin conformation at the upstream and promoter region of <i>OsDREB1b</i> in the transcription “off” state. During cold stress, changes in region specific histone modification marks promote the alteration of chromatin structure to facilitate the binding of transcription machinery for proper gene expression.</p></div

Change in RNA polymerase and nucleosome occupancy during cold stress.

<p>The relative enrichment of RNA Pol II binding at the promoter and coding region of (A) <i>OsDREB1b</i> and (B) <i>OsDREB2a</i> was determined by ChIP assay using antibody against RNA Pol II CTD (8WG16). The ChIP data was normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s <i>t</i> test and the significant changes (P≤0.05) were marked by *.</p

Change in chromatin structure at the promoter and upstream region of <i>OsDREB1b loci</i> using micrococcal nuclease digestion.

<p>MNase accessibility in control and cold stress treated nuclei (2 Hr and 4 Hr) was detected with quantitative PCR based method. Nuclei were digested with MNase (30 U/ml) for increasing time period as indicated. The isolated DNA was used for PCR reaction with primers specific for promoter and upstream region. The amount of DNA amplified at each time point was normalised to that at time 0 and plotted against time to compare the rate of degradation. The data represented here is a mean of three independent experiments with standard error bars and statistically significant values were marked with *. (A and D) rate of degradation for actin promoter; (B, C, D and E) <i>OsDREB1b locus</i>.</p

Expression profile of <i>OsDREB1b</i> and <i>OsDREB2a</i> gene under cold and high salt stress condition.

<p>The transcript of <i>OsDREB1b</i> and <i>OsDREB2a</i> gene was monitored by northern blot analysis using gene specific probes generated from 3′ end of the gene. The rice actin gene (OSJNBa0005K07) was used as internal control.</p

Alteration of histone H3 modifications during cold stress.

<p>Relative change in Histone H3 acetylation (H3K9ac, H3K14ac, and H3K27ac) during cold stress at (A) region Ia (−232 to −40) and region III (+157 to +307) (B) region Ib (−415 to −246) and region II (−610 to −440) of <i>OsDREB1b</i> gene. (C) Relative change in histone modifications at promoter and upstream region of <i>OsDREB2a</i> during cold stress. Samples were analysed by real time PCR except (A). The mean values for each region were normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s <i>t</i> test and the significant changes (P≤0.05) were marked by *. (C) Western blot showing H3K9ac, H3K14ac and H3K27ac signal in whole cell extract isolated from control and cold stress treated rice seedlings.</p

Relative fate of <i>OsDREb1b</i> transcription and histone modifications during cold exposure and subsequent return to normal growth temperature.

<p>17 days old rice seedlings were subjected to cold stress and then recovered to normal growth temperature for 6(A) Comparison of <i>OsDREB1b</i> transcript level when plants were returned to normal temperature after cold stress. (B, C and D) Comparison of histone modifications at the <i>OsDREB1b</i> locus during recovery after cold stress. The data represented here is a mean of three independent experiments with standard error bars. Statistically significant values were marked with *.</p

Sequences of the PCR primers.

<p>Sequences of the PCR primers.</p

STAT3 silencing potentiate Ara-LAM mediated abrogation of SOCS3 expression in <i>Leishmania-donovani</i> infected macrophages.

<p><i>Peritoneal macrophages (2×10⁶ cells/mL) were transfected with control siRNA or STAT3-specific siRNA, subsequently followed by Ara-LAM treatment (for 3 hr) and Leishmania infection for 24 hr. The cells were then lysed and subjected to Western blot with anti-SOCS3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (A). In a separate experiment macrophages were transfected with either control-siRNA or</i> STAT3 <i>specific siRNA, subsequently followed by Ara-LAM treatment for 3 hr and Leishmania infection for another 3 h. RNA was isolated and semi quantitative RT-PCR analyses for SOCS3 and GAPDH were done. Data represented here are from one of three independent experiments, all of which yielded similar results (B). Changes in expression of SOCS3 mRNA were also determined by quantitative real-time PCR. Results are presented as changes (n-fold) relative to uninfected control cells. The experiment was repeated 3 times, yielding similar results (C). *P<.001 compared with infected macrophages. The comparison of SOCS3 expression between Ara-LAM treated infected macrophages and STAT3 siRNA group did not show any statistical significance.</i></p

Ara-LAM mediated effector function depends on the reciprocal activation of p38MAPK and ERK–1/2.

<p><i>Peritoneal macrophages (2×10⁶cells/mL) were treated with SB203580 (SB) or PD098059 (PD) for 2 h, followed by Ara-LAM treatment for 3 hr. The cells were then infected with Leishmania parasite for 24 h and assayed for the levels of IL-12 (A) and IL-10 (C) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. Macrophages cultured in a 24-well plate (1×10⁶ cells/mL) were pretreated and infected as described above and assayed for the levels of extracellular NO as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024141#s4" target="_blank">Materials and Methods</a> (E). Asterisks indicate statistically significant induction of nitrite generation, compared with Ara-LAM–pretreated infected macrophages. *P<.001. From a separate set of cells (2×10⁶ cells/mL) RNA was isolated and levels of mRNA expression for IL-12p40 (B), IL-10 (D), inducible nitric oxide synthase 2 (iNOS2) (F) were determined by quantitative RT-PCR. Results are presented as changes (nfold) relative to uninfected control cells. The data represent the mean values</i>±<i> standard deviation of results from 3 independent experiments that all yielded similar results. In a separate experiment, the macrophages were cultured in coverglasses treated with SB or PD for 2 h, subsequently followed by 3 hr of Ara-LAM treatment and 4 h of Leishmania infection. After indicated time of incubation intracellular parasite number were assessed as described in methods. Pretreatment with SB significantly inhibited Ara-LAM –mediated parasite killing compared with levels in corresponding Ara-LAM pretreated infected controls (G). *P<.001 for SB.</i></p