Abstract
BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin.
Highlights
BCL6 is a transcriptional repressor, which plays important roles in normal B-cell development and in lymphomas
A 198 bp probe containing the CCCTC-binding factor (CTCF) binding site at BCL6 exon1A was generated by PCR amplification and incubated with nuclear extracts from HEK293T cells transfected with CTCF expression vectors
BCL6 is highly regulated during B cell differentiation such that naïve B-cells and terminally differentiated plasma cells do not express the protein whilst germinal centre B-cells express large amounts[43]
Summary
BCL6 is a transcriptional repressor, which plays important roles in normal B-cell development and in lymphomas. BCL6 is required for germinal centre formation and Tdependent antibody responses[1] (reviewed in ref.2) and must be down-regulated for terminal differentiation to plasma cells to occur[3,4]. CTCF was recently shown to bind unmethylated CpG islands within BCL6 intron 1 in a plasma cell line that does not express BCL617. In a BCL6 expressing Burkitt’s lymphoma cell line there was less CTCF binding at these intronic sites associated with increased methylation[17]. The aim of the present study was to analyse the novel CTCF binding site found in a CpG island in BCL6 exon1A and its possible biologic and mechanistic roles in BCL6 regulation in either BCL6 expressing or non-expressing cell lines and in primary cells from patients with different lymphoid neoplasms
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