The precursors of Euplotes raikovi pheromones Er‐2 and Er‐10 have been structurally characterized from the sequences of their coding regions that were amplified and cloned using the polymerase chain reaction and oligonucleotide primers corresponding to conserved sequences of the gene for phermone Er‐1. The predicted amino acid sequences contain 75 residues distributed through three domains: signal peptide, pro segment and mature pheromone. Despite the conservation of the overall length, there is variation in the size of the pro segments and of the mature pheromones. The comparison of the sequences shows a gradient of identity from the amino to the carboxyl terminus; the signal sequences are identical (with > 95% identity in the nucleotide sequences), the pro segments more variable and the mature pheromones quite diverse. The processing site of the pro pheromones, to produce the mature forms, is apparently characterized by the unusual Xaa‐Asp sequence.
Abstract Most organisms oppose many environmental stresses by rapidly enhancing synthesis of the highly conserved Hsp70 family of heat‐shock proteins. Two ciliates which are endemic in Antarctic coastal seawater, Euplotes focardii and E. nobilii , and behave as psychrophile and psychrotroph micro‐organisms, respectively, revealed a divergence in the capacity to respond to thermal stress with an activation of the transcription of their hsp70 genes. In both species, these genes were shown to be represented by thousands of copies in the cell’s somatic functional nucleus (macronucleus). However, while a strong transcriptional activity of hsp70 genes was induced in E. nobilii cells transferred from 4 to 20 °C, a much smaller increase was revealed in heat‐shocked cells of E. focardii . These findings suggest a closer adaptation to the stably cold Antarctic waters in the genetic response of E. focardii to thermal stress.
Autoimmune result from the interaction between genetic and environmental factors. Previous studies have demonstrated the implication of DNA methylation alterations in many autoimmune diseases.1 Sjögren’s Syndrome (SS) is a prototypic systemic autoimmune disease that can be primary or associated with other systemic connective tissue diseases. DNA methylation is almost exclusively found in the context of the dinucleotide sequence CpG. Methylation of regulatory sequences can lead to gene silencing and the interest in DNA methylation has been raised through multiple studies demonstrating its potential as biomarker containing valuable information for diagnosis, classification and prognosis of disease. Until now, very few data are available in pSS.
Objectives
To date, no treatment has proven effective in SS. The identification of differentially methylated regions could provide information on novel key players involved in the pathogenesis of pSS and new targets for therapeutic intervention in the future.
Methods
We analyzed genome-wide DNA methylation patterns in FACS sorted B and T-lymphocytes from 12 SS patients and 12 controls using the Illumina 450K Infinium Human Methylation 450K BeadChip monitoring quantitatively more than 480,000 CpG positions. Data was analyzed using a newly developed preprocessing pipeline for 450K data using an original subset quantile normalization approach that performs both sample normalization and efficient Infinium I/Infinium II shift correction.2 Differentially methylated regions of interest are validated in an additional set of 12 SS patients and 12 controls as well as salivary gland biopsies using pyrosequencing.
Results
1537 probes associated with 993 genes were differentially methylated between patients and controls in B lymphocytes, and 1129 probes associated with 723 genes were differentially methylated in T lymphocytes including genes from the MHC and genes associated with other autoimmune diseases. Pathway analysis showed a highly significant overlap of the genes identified in B lymphocytes with rheumatoid arthritis (p<10-8) and lupus associated genes (p<10-6) including SLC15A4 and IKFZ1, lymphomagenesis and regulation of apoptosis. Replication is currently ongoing, but the first genes replicated well in an independent biological samples. Further, comparing our data to genes found differentially methylated in synviocytes from rheumatoid arthritis (RA) patients we show that the RA-signature classified reasonably well the B cell samples into pSS patients and controls. Although the separation was not perfect (as expected as different tissues were analyzed), these results raise the hypothesis of a common DNA methylation signature of autoimmune diseases.
Conclusions
Genome-wide DNA methylation profiling identified widespread epigenetic deregulation which provide novel insight in the disease pathology and raises the possibility of a common epigenetic deregulation in AIDs.
References
Myrtue Nielsen, H. and J. Tost (2012) Epigenetic changes in inflammatory and autoimmune diseases, Subcellular Biochemistry, 61, 455-78. Touleimat, N., and J. Tost (2012) A complete pipeline for Infinium Methylation 450K BeadChip data processing using subset quantile normalization for accurate DNA methylation estimation, Epigenomics, 4, 325-41.
Acknowledgements
This work was supported by the Agence Nationale pour la Recherche (BLAN 2010 R11035LL).
ABSTRACT The ciliate Euplotes raikovi produces a family of diffusible signal proteins (pheromones) that function as prototypic growth factors. They may either promote cell growth, by binding to pheromone receptors synthesized by the same cells from which they are secreted (autocrine activity), or induce a temporary cell shift from the growth stage to a mating (sexual) one by binding to pheromone receptors of other, conspecific cells (paracrine activity). In cells constitutively secreting the pheromone E r -1, it was first observed that the expression of the E r -1 receptor “p15,” a type II membrane protein of 130 amino acids, is quantitatively correlated with the extracellular concentration of secreted pheromone. p15 expression on the cell surface rapidly and markedly increased after the removal of secreted E r -1 and gradually decreased in parallel with new E r -1 secretion. It was then shown that p15 is internalized through endocytic vesicles following E r -1 binding and that the internalization of p15/E r -1 complexes is specifically blocked by the paracrine p15 binding of E r -2, a pheromone structurally homologous to, and thus capable of fully antagonizing, E r -1. Based on previous findings that the p15 pheromone-binding site is structurally equivalent to E r -1 and that E r -1 molecules polymerize in crystals following a pattern of cooperative interaction, it was proposed that p15/E r -1 complexes are internalized as a consequence of their unique property (not shared by p15/E r -2 complexes) of undergoing clustering.
