DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells

We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation

Groundnut

Groundnut, a crop rich in nutrients, originated in South America and spread to the rest of the world. Cultivated groundnut contains a fraction of the genetic diversity present in their closely related wild relatives, which is not more than 13 %, due to domestication bottleneck. Closely related ones are placed in section Arachis , which have not been extensively utilized until now due to ploidy differences between the cultivated and wild relatives. In order to overcome Arachis species utilization bottleneck, a large number of tetraploid synthetics were developed at the Legume Cell Biology Unit of Grain Legumes Program, ICRISAT, India. Evaluation of synthetics for some of the constraints showed that these were good sources of multiple disease and pest resistances. Some of the synthetics were utilized by developing ABQTL mapping populations, which were screened for some biotic and abiotic constraints. Phenotyping experiments showed ABQTL progeny lines with traits of interest necessary for the improvement of groundnut

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Isolation and seroprevalence of Aeromonas spp. among common food animals slaughtered in Nagpur, Central India

Aeromonads are ubiquitous foodborne pathogens with a global distribution. Animal-origin foods and contaminated animals are the main sources of Aeromonas infection to humans. So far little is known about the occurrence of Aeromonas spp. in food-producing animals in India. The present study was conducted to determine the prevalence and seroprevalence of Aeromonas species from 50 each of meat, blood, and sera samples collected from cattle, buffaloes, goats, and pigs slaughtered in and around Nagpur, Central India. Alkaline peptone water and ampicillin dextrin agar were used to isolate Aeromonas spp. An indirect enzyme-linked immunosorbent assay (ELISA) was standardized by use of whole-cell antigen (WC) and outer membrane protein (OMP) of Aeromonas hydrophila (MTCC 646). Aeromonads were isolated from 44 (22%) of the meat samples, and 1 (0.5%) from the blood samples. Seroprevalence by indirect ELISA-based WC antigen was estimated as 68% in cattle, 44% in buffaloes, 60% in goats, and 30% in pigs. OMP-based ELISA yielded a seroprevalence of 56%, 48%, 52%, and 22% in cattle, buffaloes, goats, and pigs, respectively. The results revealed that OMP-based ELISA and WC-based ELISA were in agreement with one another. Isolation along with high seropositivity demonstrates the presence of foodborne Aeromonas spp. in the Nagpur city of Central India

CpG-Free Silencing Is Associated with Lack of Acetylation of H3 and H4, Lack of Methylation of H3-K4, and Replication in Late S Phase

<div><p>(A) Histone modification analysis by ChIP assays. The histograms summarize the results of quantitative PCR measurements of abundance of the EGFP coding sequence, the hβ-globin promoter, the mouse β-major promoter region (mBmaj), and a fragment of the mouse amylase coding sequence (mAmylase) in chromatin from cells containing cassette 23-β-EGFP at RL4 precipitated with polyclonal antibodies against acetylated H3-tails (left), acetylated H4-tails (middle), and methylated H3-K4-tails. The enrichment of specific sequences in modified chromatin compared to total chromatin was calculated as described in Materials and Methods. Both the EGFP coding sequence and the β-globin promoter of the CpG-free 23-β-EGFP cassette are enriched when acetylated on H3 and H4 and H3-K4 methylation and when they are in the P orientation, but not when they are in the N orientation.</p><p>(B) ChIP analysis of the CpG-containing cassette inserted at RL5 in the N or P orientation. The two orientations are similar, and, as at RL4, the enrichment is higher for the EGFP coding sequences than for the promoter.</p><p>(C) Timing of replication analysis: The histograms summarize the results of quantitative PCR measurements of the abundance of the CpG-free and CpG-containing EGFP coding sequence in FACS-separated, BrdU-immunoprecipitated newly replicated DNA fragments from cells containing the CpG-containing (left) or the CpG-free (right) LCR-β-EGFP cassettes. Both cassettes replicate early when they are in the P orientation and late when they are in the N orientation. Controls were as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g003" target="_blank">Figure 3</a>.</p><p>N, non-permissive orientation; P, permissive orientation.</p></div

Cre Recombination Per Se Does Not Lead to Silencing

<div><p>Cells containing cassette 234-β-EGFP at RL5 were transfected with the Cre recombinase, and inverted sub-clones were identified using primer pairs p1/p2 (specific for the P orientation) and p1/p3 (specific for the N orientation).</p><p>(A) Location of the primers used to identify the inverted clones.</p><p>(B) P to N inversion: The histogram on the left illustrates expression profile of a clone with insertion in the P orientation before inversion. The histogram on the right illustrates expression 8 wk post-inversion. The ethidium bromide stain illustrates the PCR analysis demonstrating the inversion.</p><p>(C) Same as (B) but for the N to P inversion. This analysis clearly demonstrates that Cre recombination per se does not lead to silencing (see text). As reported previously, levels of expression in the N orientation at RL5 are lower than in the P orientation [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-b032" target="_blank">32</a>].</p><p>N, non-permissive orientation; P, permissive orientation.</p></div

Inversion of the CpG-Free Cassette and Analysis of Chromatin Structure of the Inverted Cassette

<div><p>(A) Southern blots demonstrating inversion of the 23-β-EGFP cassette from the N to the P orientation. N to P lanes, Hind III digested genomic DNA from three N to P inverted sub-clones isolated after transfection of cells with cassette CpG-free 23-β-EGFP in the N orientation with a Cre recombinase expression plasmid. N and P, control sub-clones.</p><p>(B) FACS analysis of representative clones with the CpG-free and CpG-containing cassettes in the P or N orientation or after N to P inversion. <i>x</i>-axis, forward scatter; <i>y</i>-axis, EGFP fluorescence. N to P inversion leads to the reactivation of the CpG-free but not the CpG-containing cassette.</p><p>(C) Quantitative RT-PCR analysis of EGFP expression on total RNA extracted from two to five sub-clones with the CpG-containing or the CpG-free cassette in the P or N orientation and after N to P in vivo inversion. Results are expressed as percent of β-2-micro-globulin expression (100 × number of EGFP transcripts/number of B2M transcripts). This analysis confirms the FACS data and demonstrates that silencing and reactivation of the CpG-free cassette occurs primarily at the transcriptional level.</p><p>(D) Plots illustrating replication timing analysis of the CpG-free cassette after N to P inversion. The graphs and controls are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g003" target="_blank">Figure 3</a>C. After N to P inversion, the CpG-free cassette replicates early in S phase.</p><p>(E) ChIP analysis of the CpG-free 23-β-EGFP cassette. The graphs and controls are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g003" target="_blank">Figure 3</a>B. The results show that the CpG-free cassette is enriched in H3 acetylation and poor in H3-K9 tri-methylation in the P orientation, but that it is poor in H3 acetylation and rich in H3-K9 tri-methylation in the N orientation. After N to P inversion, the CpG-free cassette becomes enriched in H3 acetylation but poor in H3-K9 tri-methylation.</p><p>N, non-permissive orientation; P, permissive orientation.</p></div

Analysis of the Chromatin Structure of the CpG-Containing Cassette

<div><p>(A) Bisulfite sequencing analysis was performed on cells with CpG-containing cassette 234-β-EGFP at RL4 after N to P inversion. There were 11 molecules sequenced. Most of the CpGs were methylated (see text).</p><p>(B) Histograms illustrating H3 acetylation and H3-K4 methylation results on the CpG-containing cassettes. The analysis was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g002" target="_blank">Figure 2</a>. Mouse β-major globin (mBmaj) is a positive control for H3 and H4 acetylation and a negative control for H3-K9 tri-methylation. The α subunit of the guanine nucleotide-binding protein (mGnasx) and the mouse amylase genes are a positive control for H3-K9 tri-methylation and negative controls for H3 acetylation. The CpG-containing cassette is enriched in H3 acetylation and H3-K4 methylation in the P orientation but poor in acetylated H3 and H3-K4 methylation. After N to P inversion, the cassette remains poor in H3 acetylation and H3-K4 methylation.</p><p>(C) Plots illustrating replication timing analysis of the CpG-containing cassette after N to P inversion. The analysis was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g002" target="_blank">Figure 2</a>. Mouse β-major globin and amylase are early and late replicating controls. After N to P inversion, the CpG-containing cassette replicate late in S phase.</p><p>N, non-permissive orientation; P, permissive orientation.</p><p>(D) Histograms illustrating H3-K9 tri-methylation results on the CpG-containing cassettes. The analysis was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g002" target="_blank">Figure 2</a>. The controls are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020065#pgen-0020065-g003" target="_blank">Figure 3</a>B. The enrichment is low when the cassette is in the P orientation and high in the N orientation. The enrichment remains high after N to P inversion.</p></div