Immune parameters that are known to play a major role in genetic resistance to MDV are correlated with innate immune responses, such as NK cell activity, production of nitric oxide and cytokines, such as, interferons. Recent studies have identified host cytokines such as IL-18 and IFN-γ that contribute to the initiation and continuation of latency
. However, cytokine levels can undergo rapid flux in response to infection, and consistent with this, we did not observe any epigenetic changes associated with these genes in the MHC-congenic lines used in our study (Additional file
1: Figure S8). This suggests the existence of other extrinsic factors responsible for transcriptional variations between resistant and susceptible chickens at this stage of the disease.
A global comparison of histone modification levels in the two inbred chicken lines yielded some interesting results. As expected, a majority of SERs were found in all the experimental groups, indicating a high level of epigenetic similarity between the lines in addition to inherent genetic similarity. In the case of H3K27me3, the percentage of ubiquitous SERs was relatively low (23.4%), although this was likely due to lower precision of the peak caller for broad chromatin marks. Besides, most of the SERs detected in a subset of samples corresponded to regions of low enrichment, which may also be the reason behind the relatively low number of differential SERs detected in our study.
Genes that have been previously associated with MD and various other cancers showed differential marks that are either MD-induced (MX1, CTLA-4, EAF2 and GAL) or line-specific (IGF2BP1 and MMP2). The increase in H3K4me3 marks around the TSS of MX1, a gene with known antiviral properties, appeared to be correlated with upregulated expression in both lines in response to MDV infection. However, lowered overall mRNA levels in the resistant line suggest additional factors could be involved in the regulation of this gene. Similarly, increased mRNA levels of the lymphocyte surface marker CTLA4 is possibly due to the presence of larger numbers of T cells in MDV infected birds as a result of higher levels of infection. EAF2 functions as an apoptosis inducer in addition to being a tumor suppressor, and therefore, its downregulation could contribute to higher tumor incidence rates in line 72. However, it is not clear why a significant increase in H3K27me3 levels did not have any effect on transcript levels in the resistant line.
IGF2BP1 is believed to act by stabilizing the mRNA of the c-myc oncogene and therefore, the higher expression of this gene and a more stable c-myc gene product might play a role in increasing MD susceptibility in line 72 birds via increased cell proliferation and transformation. The matrix metalloprotease MMP2 is upregulated after infection in the resistant line 63, similar to the previously observed increase at the neoplastic stage of MD. However, mRNA levels were similar in the two lines before infection contrary to the difference in H3K4me3 levels suggesting that additional factors are responsible for regulating this gene.
The correlation between observed differential histone marks and transcript levels was moderate at best. Indeed, differential H3K4me3 marks were strongly predictive of gene expression levels but the correlation between H3K27me3 and mRNA levels was relatively poor. There could be various reasons for this – indeed, H3K27me3 levels had a non-linear relationship with gene expression with higher marks showing little difference in the effect on expression. Therefore, in this tissue, the levels of H3K27me3 may not be a very good indicator of gene expression levels. Also, the transcription of these genes might be controlled by other factors with the change in H3K27me3 levels only incidental.
Bivalent domains were detected on transcriptional regulators BCL6, CITED2 and EGR1 and the galanin receptor GALR1. The epigenetic and transcriptional signatures observed on these genes indicated that they were poised at the latent stage of the disease, but with crucial differences in the two lines. Increased repressive marks in the susceptible line correlated with significant downregulation of the genes, while in line 63, the increase in both marks appeared to compensate for each other with no eventual effect on gene transcription. This suggests that some ‘poised’ bivalent genes can become highly repressed even with a relatively small increase in H3K27me3 marks. The change in the chromatin levels could also be correlated with an increase in cell populations having the repressive mark. Taken together, the above findings point towards the existence of finely balanced epigenetic control of transcription, which may be necessary to mount a rapid response by the immune system. However, this machinery could potentially be hijacked by a pathogen and result in an aberrant phenotype. The effect of MDV infection on the bivalent domain on GALR1, in particular, suggests the repression and potential loss of its anti-proliferative effects. Thus, the galanin system possibly plays an important and hitherto unknown role in MD progression and could be a novel target for long-term control of the disease.
One of the major players in MDV-induced malignant transformation is Meq, a virus-encoded oncoprotein that has diverse functions, e.g. transactivation, chromatin remodeling and regulation of transcription. Meq interacts with and sequesters the tumor suppressor protein p53, resulting in the dysregulation of cell-cycle control
 and inhibition of the transcriptional and apoptotic activities of the protein
. Several genes that show epigenetic changes in response to MDV infection have been associated with p53. Downregulation of CITED2 stabilizes the p53 protein leading to its accumulation
. The BCL6 gene product is believed to contribute to lymphomagenesis by inactivation of p53
. Besides, EAF2 has also been shown to interact with and suppress the function of p53
. The downregulation of all of the above genes in susceptible birds after MDV infection points towards the increased production of p53 and a robust anti-tumor response. That we still observe higher tumor incidence rates in this line, suggests the presence of large amounts of inactivating viral Meq protein which, in turn, indicates that increased numbers of MD-infected cells are present in the susceptible line at this stage of the disease. A majority of tumors are believed to result from the viral transformation of CD4+ T cells some of which are infected at the latent stage of MD
. The larger number of virus-infected cells produced in the susceptible line is possibly due to lowered immunocompetence as a result of the early stages of infection. Thus, a more detailed investigation of the early cytolytic stage of MD is necessary to shed further light on the causes behind the divergent response to MD observed in these birds.
Whole tissues represent a mixture of various cell populations, and observed epigenetic changes might be due to a change in chromatin marks in a particular cell type or a variation in the relative number of cells carrying active or repressive histone marks. However, such in vivo studies are representative of the host response at a systems level wherein different cell types might interact in a cooperative manner to fight infection. Thus, while the study of pure cell populations is likely to yield greater discriminative power, the investigation of tissue macroenvironments is, perhaps, closer to reality.
This study focused on the thymus tissue as it is a major immune organ and contains a significant population of T lymphocytes in various stages of differentiation. Our earlier study of the MDV-induced transcriptome in these birds indicated the presence of line-specific differences at the latent stage of MD
. In addition, birds susceptible to MD suffer thymic atrophy during the early stages of infection
, indicating the importance of understanding changes in this organ to elucidate the mechanisms involved in disease progression. Ongoing studies in our lab include other tissues, e.g. spleen
, and a time-course through the various stages of infection, to further investigate the systemic effects of MD and the epigenetic basis of MD resistance.