Identification of Cyclin D3 as a Direct Transcriptional Target of E2A Using Damid.
Authors: Choi John K. , Song Siyuan , Cooperman Jonathan , Letting Danielle L. , Blobel Gerd A. 
Published: October 2019

Abstract
The transcription factor E2A is required for very early B cell development. The exact mechanism by which E2A promotes B cell development is unclear and cannot be explained by the known E2A targets, components of the pre-B cell receptor and cyclin dependent kinase inhibitors, indicating additional pathways and targets remain to be identified. We had previously reported that E2A can promote precursor B cell expansion, promote G1 cell cycle progression, and induce the expressions of multiple G1 phase cyclins including cyclin D3, suggesting that E2A induction of these genes may contribute to early B cell development. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated a luciferase reporter plasmid containing the 1kb promoter of cyclin D3 that contains two consensus E2A binding sites (E-boxes); however, deletion of the E-boxes did not disrupt the transactivation by E2A. We hypothesized three possible mechanisms: 1) indirect activation of cyclin D3 via another transcription factor, 2) binding of E2A to cryptic non-E-boxes, or 3) recruitment of E2A to the promoter via interaction with other DNA binding factor. To test the first possibility, promoter occupancy was examined using the DamID approach. In this approach, a fusion protein consisting of E. coli DNA adenosine methyltransferase (DAM) and a transcription factor of interest is expressed at low levels, resulting in specific methylation of adenosine residues within 2–5 kb of the transcription factor target sites. A fusion construct composed of E2A and DAM (E47Dam), was subcloned in lentiviral vectors, and used to transduce precursor B cell lines. The methylated adenosine residues were detected using a sensitive ligation-mediated PCR (LM-PCR) assay that required only 1 ug of genomic DNA and can detect methylation even if only 3% of the cells express E47Dam; no methylated adenosines were detected in control cells, indicating that all methylated residues resulted from E47Dam. Specific adenosine methylation was identified at the IgH intronic enhancer, a known E2A target site, but not at the non-target sites, CD19, HPRT, and GAPDH promoters. Specific methylation was detected at the cyclin D3 promoter but not 10 kb down-stream, despite similar concentrations of E-boxes at both sites. Chromatin immunoprecipitation analysis confirmed the DamID findings and further localized the binding to within 1 kb of the two E-boxes in the cyclin D3 promoter. To distinguish between the two remaining mechanisms (cryptic non-E-boxes versus recruitment via other DNA binding factors), two point mutations were introduced into E47Dam that disrupted its DNA binding activity. The mutated E47Dam continued to methylate at the cyclin D3 promoter. We conclude that E2A can be recruited to the cyclin D3 promoter, independent of E-boxes or E2A DNA binding activity. Our findings raise the possibility that some direct E2A target genes may lack functional E-boxes. Furthermore, mutated E2A, lacking an E2A DNA binding domain, that is seen in 6% of pediatric ALLs may still activate a subset of E2A target genes. Finally, our application of lentiviral vectors and LM-PCR to the DamID approach should permit analysis of primary human precursor B cells, despite the limitations in cell number and transduction efficiency.