The B cell transcription program mediates hypomethylation and overexpression of key genes in Epstein-Barr virus-associated proliferative conversion

Genome biology (Impact Factor: 10.81). 01/2013; 14(1):R3. DOI: 10.1186/gb-2013-14-1-r3
Source: PubMed


Epstein-Barr virus (EBV) infection is a well characterized etiopathogenic factor for a variety of immune-related conditions, including lymphomas, lymphoproliferative disorders and autoimmune diseases. EBV-mediated transformation of resting B cells to proliferating lymphoblastoid cells occurs in early stages of infection and is an excellent model for investigating the mechanisms associated with acquisition of unlimited growth.

We investigated the effects of experimental EBV infection of B cells on DNA methylation profiles by using high-throughput analysis. Remarkably, we observed hypomethylation of around 250 genes, but no hypermethylation. Hypomethylation did not occur at repetitive sequences, consistent with the absence of genomic instability in lymphoproliferative cells. Changes in methylation only occurred after cell divisions started, without the participation of the active demethylation machinery, and were concomitant with acquisition by B cells of the ability to proliferate. Gene Ontology analysis, expression profiling, and high-throughput analysis of the presence of transcription factor binding motifs and occupancy revealed that most genes undergoing hypomethylation are active and display the presence of NF-κB p65 and other B cell-specific transcription factors. Promoter hypomethylation was associated with upregulation of genes relevant for the phenotype of proliferating lymphoblasts. Interestingly, pharmacologically induced demethylation increased the efficiency of transformation of resting B cells to lymphoblastoid cells, consistent with productive cooperation between hypomethylation and lymphocyte proliferation.

Our data provide novel clues on the role of the B cell transcription program leading to DNA methylation changes, which we find to be key to the EBV-associated conversion of resting B cells to proliferating lymphoblasts.

Download full-text


Available from: Claire Shannon-Lowe, Oct 07, 2015
1 Follower
27 Reads
  • Source
    • "A sudden change in the cell cycle/division status of the cell, including transformation from a quiescent to proliferative status in aberrant context, may result in inefficient maintenance of the epigenetic marks. We have also observed a similar behavior when focusing on DNA methylation during the conversion of resting to proliferating B cells (34), that is decreased in genes in euchromatic regions, particularly those regulated by B cell specific factors. In this context of inefficient maintenance, different elements of the epigenetic and nuclear organization machinery that are normally coupled may find their links loosened. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.
    Nucleic Acids Research 10/2013; 42(1). DOI:10.1093/nar/gkt886 · 9.11 Impact Factor
  • Source
    • "This method, aiming at the amplification of unmethylated Alus (AUMA), was performed as described [31,39]. Products were resolved on denaturing sequencing gels. "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA methylation is a key epigenetic mechanism for driving and stabilizing cell-fate decisions. Local deposition and removal of DNA methylation are tightly coupled with transcription factor binding, although the relationship varies with the specific differentiation process. Conversion of monocytes to osteoclasts is a unique terminal differentiation process within the hematopoietic system. This differentiation model is relevant to autoimmune disease and cancer, and there is abundant knowledge on the sets of transcription factors involved. Here we focused on DNA methylation changes during osteoclastogenesis. Hypermethylation and hypomethylation changes took place in several thousand genes, including all relevant osteoclast differentiation and function categories. Hypomethylation occurred in association with changes in 5-hydroxymethylcytosine, a proposed intermediate toward demethylation. Transcription factor binding motif analysis revealed an overrepresentation of PU.1, NF-kappaB and AP-1 (Jun/Fos) binding motifs in genes undergoing DNA methylation changes. Among these, only PU.1 motifs were significantly enriched in both hypermethylated and hypomethylated genes; ChIP-seq data analysis confirmed its association to both gene sets. Moreover, PU.1 interacts with both DNMT3b and TET2, suggesting its participation in driving hypermethylation and hydroxymethylation-mediated hypomethylation. Consistent with this, siRNA-mediated PU.1 knockdown in primary monocytes impaired the acquisition of DNA methylation and expression changes, and reduced the association of TET2 and DNMT3b at PU.1 targets during osteoclast differentiation. The work described here identifies key changes in DNA methylation during monocyte-to-osteoclast differentiation and reveals novel roles for PU.1 in this process.
    Genome biology 09/2013; 14(9):R99. DOI:10.1186/gb-2013-14-9-r99 · 10.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clofarabine, a purine nucleoside analog, is used in the treatment of hematologic malignancies and as induction therapy for stem cell transplantation. The discovery of pharmacogenomic markers associated with chemotherapeutic efficacy and toxicity would greatly benefit the utility of this drug. Our objective was to identify genetic and epigenetic variants associated with clofarabine toxicity using an unbiased, whole genome approach. To this end, we employed International HapMap lymphoblastoid cell lines (190 LCLs) of European (CEU) or African (YRI) ancestry with known genetic information to evaluate cellular sensitivity to clofarabine. We measured modified cytosine levels to ascertain the contribution of genetic and epigenetic factors influencing clofarabine-mediated cytotoxicity. Association studies revealed 182 single nucleotide polymorphisms (SNPs) and 143 modified cytosines associated with cytotoxicity in both populations at the threshold p ≤ 0.0001. Correlation between cytotoxicity and baseline gene expression revealed 234 genes at p ≤ 3.98x10(-6). Six genes were implicated as: (1) their expression was directly correlated to cytotoxicity, (2) they had a targeting SNP associated with cytotoxicity, and (3) they had local modified cytosines associated with gene expression and cytotoxicity. We identified a set of 3 SNPs and 3 CpG sites targeting these six genes explaining 43.1% of the observed variation in phenotype. siRNA knockdown of the top three genes (SETBP1, BAG3, KLHL6) in LCLs revealed altered susceptibility to clofarabine, confirming relevance. As clofarabine's toxicity profile includes acute kidney injury, we examined the effect of siRNA knockdown in HEK293 cells. siSETBP1 led to a significant change in HEK293 cell susceptibility to clofarabine.
    Human Molecular Genetics 05/2013; 22(19). DOI:10.1093/hmg/ddt240 · 6.39 Impact Factor
Show more