Epigenetic silencing of the interferon regulatory factor ICSBP/IRF8 in human multiple myeloma.

OBJECTIVE: Multiple myeloma (MM) is presently an incurable malignant plasma cell tumor. The objective of this study was to investigate expression of the interferon regulatory factor family (IRF1-9) and the potential role of DNA methylation in silencing IRF genes in MM cell lines and purified MM cells from patients. MATERIALS AND METHODS: Using a panel of 13 human MM cell lines and purified CD138+ cells from nine MM patients, expression of IRF genes was investigated by quantitative reverse transcriptase polymerase chain reaction and Western blot. DNA methylation of the interferon consensus sequence-binding protein (ICSBP/IRF8) gene was measured using pyrosequencing, and the effect of promoter methylation on expression was analyzed by in vitro methylation of a cloned ICSBP/IRF8 promoter, and treatment of MM cells with 5-aza-2'-deoxycytidine (DAC). RESULTS: Eight of thirteen of the MM cell lines were found to lack ICSBP/IRF8 expression, associated with hypermethylation of the CpG island in the ICSBP/IRF8 promoter. We also found that ICSBP/IRF8 was significantly underexpressed in primary MM cells, whereas the ICSBP/IRF8 promoter was methylated in only one of nine of primary purified CD138+ MM samples. DAC-mediated demethylation restored endogenous ICSBP/IRF8 expression, whereas in vitro methylation silenced the promoter. CONCLUSION: Expression of the ICSBP/IRF8 gene is silenced in a majority of MM cell lines and primary CD138+ MM cells. DNA methylation of the ICSBP/IRF8 gene is a frequent event in MM cell lines, but silencing is also observed in the absence of methylation. These results suggest that silencing of ICSBP/IRF8 expression, by DNA methylation or other epigenetic mechanisms, may be associated with the malignant phenotype of MM.

Positive histone marks are associated with active transcription from a methylated ICSBP/IRF8 gene.

Epigenetic modifications are critical for regulating many different aspects of normal cell biology and tumourigenesis. Gene expression may be epigenetically silenced by DNA-methylation and histone modifications, resulting in remodelling of chromatin into a repressed state. We performed DNA-methylation analysis of the ICSBP/IRF8 gene, a member of the IRF family of transcriptional regulators expressed in monocytic and lymphocytic cells, in human monoblastic U-937 cells. We found complete methylation of all 39 CpG positions located in a 308 bp sequence encompassing the proximal promoter and transcriptional start site of the ICSBP/IRF8 gene. However, strikingly, the ICSBP/IRF8 gene is still expressed. Chromatin Immuno-precipitation (ChIP) showed that RNA-Polymerase II was present at the major transcriptional start site. Investigating the histone modifications across the ICSBP/IRF8 gene we found the positive histone marks H3K9ac and H3K4me3 to be enriched at the promoter, whereas the level of H3K9me3 was low. This suggests that an active chromatin structure, indicated by histone H3 modifications and enrichment for RNA Pol II, can over-ride the silencing effect of DNA-methylation at the promoter, thereby permitting transcription of the ICSBP/IRF8 gene.

Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.

Regulation of the expression of prtW::gusA fusions in Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora, a Gram-negative phytopathogenic bacterium, secretes an extracellular metalloprotease, PrtW. Previous results demonstrated that protease activity is necessary for the normal progression of disease symptoms caused by this bacterium. The present study revealed that the prtW gene constitutes an independent transcriptional unit. It is demonstrated that introduction of the prtW(+) plasmid in trans into the prtW(-) mutant restores the protease activity in this strain. Gene fusions to the gusA (beta-glucuronidase) reporter were employed to analyse the transcription of prtW. The transcription of prtW is dependent on many environmental signals. When the bacteria were grown in the presence of potato extract, the expression of the protease gene was markedly higher at the beginning of the exponential phase of growth than that observed when cells were grown in the presence of polygalacturonate (PGA). Analysis of the promoter revealed that an essential regulatory region resided between 371 and 245 bp 5' of the translational start site. As this sequence showed no homology to the KdgR box it may be involved in the binding of an unknown negative regulator protein in E. carotovora subsp. carotovora. The differential responses of prtW expression to potato extract and to PGA appeared to be dependent on the KdgR repressor and the response regulator ExpA. According to the results presented here, it is conceivable that the multiple regulatory network allows flexibility in the expression of the prtW gene during different stages of infection.