The rationale for examination of the epigenome in SLE is that it could provide valuable insights into gene sets with an altered competence for expression as well as the more typically defined gene sets with altered transcript levels as defined by expression arrays. Our study is the first to examine the epigenome in SLE patients and we found that the epigenome of monocytes is widely altered by the disease. Although it is possible or even likely that medications could contribute to the altered epigenome, patients were selected with minimally active disease and limited medications.
Monocytes have been extensively studied in SLE patients and exhibit abnormal behaviors (35
). Macrophage involvement in atheroma formation contributes to the accelerated atherosclerosis seen in SLE (7
) and renal macrophage infiltration is associated with a poor renal prognosis (9
). The longevity of the tissue macrophage could contribute to disease persistence and macrophage cytokine production could mold subsequent T cell responses. It is not currently known whether epigenetic changes acquired as a circulating monocyte are retained after differentiation into tissue macrophages. Retention of acquired changes to the epigenome could potentially modulate subsequent responses.
In general, H4ac was increased in SLE patients compared to controls. In order to index the data to derive biological insights, we sought for commonalities amongst over-expressed genes and increased H4ac genes. More importantly, when we sought pathways by network analysis, αIFN, NFκB and MAP kinases were consistently implicated. The proteasome network identified in the HH gene set is significant because proteasome components are known to be elevated in SLE and treatment of murine lupus models with a proteasome inhibitor was therapeutic (44
When we sought pathways by bioinformatically filtering genes by potential TFBS, three transcription factors were identified as being themselves upregulated in SLE and having binding sites over-represented upstream of both increased H4ac genes and over-expressed genes. These three transcription factors, GATA3, IRF1, and LEF1/TCF1 can be implicated in some of the features seen in SLE monocytes. Although the IRF1 binding site is identical to that of other IRF proteins, IRF1 was also over-expressed in SLE monocytes and was implicated in networking analyses. We further attempted to identify disease-specific pathways by filtering the information based on p value. Ten focus genes, identified as having both significantly increased expression and increased H4ac, had a very high enrichment of BLIMP1, IRF1, And LEF1/TCF1 binding sites in their upstream region and these binding sites colocalized with H4ac peaks. BLIMP1 is critical for macrophage differentiation and it represses c-myc, leading to cessation of cell division (46
). LEF1/TCF1 is a transcriptional regulator of the Wnt/β catenin pathway, which is important for macrophage proliferation and survival (47
). IRF1 was the most consistently implicated transcription factor in the cumulative SLE analyses. IRF1 is induced by interferons and TNFα and it acts as a weak transcriptional activator of genes involved in the antiviral response, immune modulation, and anti-tumor effects (48
). IRF1 gene deletion ameliorates renal disease in a murine lupus model, supporting a potential mechanistic role (49
). IRF1 cannot directly acetylate H4, but it can bind CBP which can then acetylate histones (50
). IRF1 is strongly induced by EBV, which has also been linked to SLE (51
). Therefore, these data provide a rationale for further investigation of IRF1 specifically and a potential explanation for the consistent finding of upregulated genes downstream of type I interferon while type I interferons themselves are inconsistently seen in patients.
SLE is clearly a complex disorder and there is likely heterogeneity in the pathologic processes. The consistency in identifying IRF1 was surprising. To further investigate the impact of the interferon pathway in SLE, we treated control monocytes with αIFN. This in vitro
model does not mimic the milieu of the host, however, it allows a simplified analysis of one variable. αIFN treatment was able to reproduce some of the gene expression and H4ac changes seen in SLE. One gene, IFIT3, was identified in the increased H4ac gene lists of both SLE and αIFN treated cells and in the increased expression gene lists of both SLE and αIFN treated cells. IFIT3 is known to be induced by type I interferons and has been demonstrated to have increased expression in both SLE and Sjogrens syndrome (52
). These data confirm an important role for αIFN, but also demonstrate the complexity of the effects on cells. αIFN effects accounted for a significant portion of the gene expression changes seen in SLE patients but a relatively small portion of the H4ac changes. This could reflect the short time frame for αIFN treatment or the limitations of an in vitro
system, however, it is likely that multiple stimuli converge on the monocyte to induce the disease-specific effects.
An important aspect of these analyses is the ability to leverage four types of data to improve the focus. In SLE, a straight comparison of genes with increased H4ac and increased expression revealed little commonality. Applying TFBS analysis revealed much more commonality. This was also true when αIFN treated cells were analyzed. The greatest enrichment of all analyses was found by analyzing TFBS overlap between increased H4ac in SLE monocytes and αIFN treated monocytes demonstrating that epigenetic studies can be powerful in identifying biological themes.
This study characterized a single histone modification using an array restricted to the promoters of annotated genes. H4ac at enhancers or other distant regulatory regions would not be identified by our study. Therefore, this study represents a minimal estimate of the full extent of the epigenetic changes seen in SLE monocytes. The importance of examining histone modifications in SLE is that they can serve to perpetuate pathologic gene expression patterns. In many cases, they are concordant with gene expression, but they can also reflect competence for expression and in so doing mold the character of a cell's behavior. These analyses confirm the importance of type I interferons and suggest that they may have a sustained effect by inducing transcription factors and by altering the epigenome.