Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells.

Eden Kleiman,Salvatore Loguercio,Ann J Feeney

doi:10.3389/fimmu.2018.02074

Abstract

To date there has not been a study directly comparing relative Igκ rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Igκ repertoire from the same pre-B cell pool. We find that ~20% of Vκ genes have rearrangement frequencies ≥2-fold up or down in RNA vs. DNA libraries, including many members of the Vκ3, Vκ4, and Vκ6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual Vκ genes rearranged at very different frequencies, and also displayed very different Jκ usage. Regression analysis revealed that the greatly unequal Vκ gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vκ genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Igκ rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of Jκ-distal Vκ genes, particularly Vκ10-96 and Vκ1-135. Regression analysis indicates that PU.1 binding is the highest predictor of Vκ gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iEκ−/− pre-B cells reveal that iEκ actively influences Vκ gene usage, particularly Vκ3 family genes, overlapping with a zone of iEκ-regulated germline transcription. These represent new roles for iEκ in addition to its critical function in promoting overall Igκ rearrangement. Together, this study provides insight into many aspects of Igκ repertoire formation.

Highlights

The ability of the B-cell receptor to recognize virtually any pathogenic epitope relies on the random nature of Ig V(D)J rearrangement to generate a vast diverse repertoire
Our data shows that ∼20% of functionally classified Vκ genes have ≥2-fold apparent differences in the frequency of rearrangements when comparing data obtained from genomic DNA (gDNA) vs. RNA repertoires in pre-B cells
We show that individual Vκ genes display extremely varied Jκ gene usage, consistent with previous data [8]

Summary

Introduction

The ability of the B-cell receptor to recognize virtually any pathogenic epitope relies on the random nature of Ig V(D)J rearrangement to generate a vast diverse repertoire. An unbiased method exists for interrogating the RNA repertoire using 5′ RACE PCR to generate a cDNA library with primers at the Cκ or Cμ exon and ligated adaptor [5, 6]. Two labs have recently developed an unbiased method for assaying gDNA rearrangements [7,8,9]. Both techniques have their respective advantages and limitations. Use of genomic DNA (gDNA) allows for an unbiased assessment of rearrangement frequencies because each cell only has two chromosomes from which rearrangements will be detected. Use of RNA examines the repertoire after transcription and could be influenced by differential promoter strengths and posttranscriptional regulation. RNA libraries predominantly assay productive rearrangements due to nonsense-mediated decay of many non-productive rearrangements [10], whereas amplification of gDNA will reveal all non-productive as well as productive rearrangements

Methods

Results

Conclusion