Methylation Regulates Expression of Novel Splice Variant and Wild Type Transcripts of IRF8.

Interferon regulatory factor 8 (IRF8) is a transcription factor that plays a critical role in normal hematopoiesis. IRF8^−/− transgenic mice develop a myeloproliferative syndrome that transforms to acute myeloid leukemia (AML). IRF8 expression varies dramatically in the blasts of AML patients. A number of biological processes, including epigenetic changes, mutations, or alternative splicing, may contribute to the variability of IRF8 expression in AML patients. We investigated the potential causes of the aberrant IRF8 expression in AML blasts.

The entire coding sequence of IRF8 was amplified using HiFi Taq polymerase and sequenced from 7 leukemic cell lines and 12 AML samples. We did not find any mutations associated with aberrant expression of IRF8.

The initial studies examining IRF8 coding region suggested transcript deviation in the 5′ region. The GeneRacer kit was used to sequence 5′-capped mRNA. We identified 3 previously-undescribed transcript variants. In all 3 sequences, exon 1 was spliced out and replaced by nucleotides from the terminal end of intron 1 (Figure 1A). Some of the splice variants contained potential in-frame start codons.

We examined the expression levels of transcript variants in 12 leukemic cell lines, 246 AML samples, and hematopoietic subpopulations of cells from “healthy” adults. IRF8-SVs were expressed at significantly higher levels in some AML blasts than normal hematopoietic cells. In fact, AML cell lines with the highest IRF8 levels primarily expressed the IRF8-SVs rather than the IRF8-WT.

Interferon response element (pIRE) within the IRF8 promoter, which controls the expression of the gene, was examined by PCR after bisulfite conversion. Cell lines with very low IRF8 expression were often methylated at the pIRE locus. In addition, some cell lines with marked over-expression of IFR8-SVs also displayed methylated pIRE, suggesting that promoter methylation may also be playing a role in controlling the expression of the splice variants. pIRE methylation was also examined in a more limited number of AML samples, finding that blasts with low expression of IRF8 were methylated at this locus (Figure 1B).

Our previous studies suggested that methylation may play a role in controlling IRF8-SVs expression. Therefore, we examined the effect of demethylating agents on IRF8-SVs expression in U937 cells – a leukemic cell line with very high levels of IRF8-SVs. IRF8 transcripts (WT and SVs) were examined before and after exposure to therapeutic levels of 5-azacytidine (5-Aza). These studies suggested that 5-Aza exposure and subsequent demethylation of the pIRE locus was associated with restoration of the IRF8-WT and decreased IRF8-SVs expression (Figure 1C). This later data provides additional evidence that pIRE hypermethylation may regulate the expression of the novel IRF8-SVs.

We have identified novel IRF8 transcript variants that are over-expressed in AML blasts. These novel IRF8-SVs introduce additional nucleotides at the 5′ region of the gene and may result in a new start codon. AML blasts with high levels of IRF8 transcript often over-express IRF8-SVs. Although the functional significance of these variants is unknown, methylation may play a role in regulating their expression, such that demethylating agents decrease IRF8-SVs and promote the IRF8-WT expression. Additional studies are planned to investigate the biology and clinical significance of IRF8-SVs. Initial studies suggests that over-expression of IRF8-SVs is associated with an inferior clinical outcome for adult AML patients.

Figure 1.

View largeDownload slide

Regulation of IRF8 Expression. A. Structure of IRF8 transcripts. Black bent arrows - start codons; splice donor/acceptor cites connected by dotted lines; pIRE methylation - CH₃; primers - black half-arrows; probe - green solid line. B. Figure shows methylation (top) and expression (bottom) of IRF8. Cells from normal donors and lymphocytes (CD3) from AML BM show no evidence of methylation of pIRE and higher expression. AML blasts (CD34) are methylated and show a significant decrease of IRF8 expression. C. Figure shows restoration of IRF8-WT and decrease of IRF8-SVs expression in U937 exposed to 5-Aza.

Figure 1.

View largeDownload slide

Regulation of IRF8 Expression. A. Structure of IRF8 transcripts. Black bent arrows - start codons; splice donor/acceptor cites connected by dotted lines; pIRE methylation - CH₃; primers - black half-arrows; probe - green solid line. B. Figure shows methylation (top) and expression (bottom) of IRF8. Cells from normal donors and lymphocytes (CD3) from AML BM show no evidence of methylation of pIRE and higher expression. AML blasts (CD34) are methylated and show a significant decrease of IRF8 expression. C. Figure shows restoration of IRF8-WT and decrease of IRF8-SVs expression in U937 exposed to 5-Aza.

Close modal

No relevant conflicts of interest to declare.