DESCRIPTION (provided by applicant): Cellular differentiation is directed by transcription factors, and it is well known that the level of mRNAs for transcription factors and other proteins change as cells differentiate. However, recent findings made possible by single cell expression analyses have revealed an unappreciated heterogeneity in transcriptional profiles of individual cells, even those of the same phenotypic stage of development. No such studies have been done to determine potential transcriptional heterogeneity among individual pro-B cells, and to determine how it changes as cells traverse the differentiation steps during pro-B cell development. Importantly, during the pro-B cell stage, Igh V(D)J recombination takes place and we make the novel hypothesis that transcriptional heterogeneity is of critical importance for the production of a diverse antibody repertoire. Non-coding germline transcription through V genes has been proposed to make genes accessible for recombination. One role of germline transcription is to mark the transcribed regions with the epigenetic mark H3K4me3, which can directly recruit Rag2. We have shown that another key role of non-coding germline transcription is to change the 3D structure of the Igh locus, bringing the transcribed region (and thus some V genes) into close proximity to E¿, the promoter of the I¿ germline transcript. E¿ is 1-2 kb from the DJ rearrangement to which one V gene will rearrange. Thus, we hypothesized that germline transcription directly results in Igh locus compaction, and we demonstrated this for the two major antisense germline transcription promoters that we identified by our RNA- seq analysis of the Igh transcriptome. However, if only the regions transcribed in these major germline transcripts are located near the DJ rearrangement, that would predict that the V genes near these regions would be more likely to rearrange than other V genes, but this is not the case. The level of other germline transcripts in the Vh portion of the Igh locus are low in general. Although it is generally believed that most functional Vh genes are transcribed at low levels in al pro-B cells, we propose a different hypothesis. Based on the emerging data from single cell transcriptional analyses, we propose that there is transcriptional heterogeneity among pro-B cells such that each pro-B cell expresses a different subset of germline transcripts, possibly influenced by differential levels of key transcription factors or possibly stochastic. If this hypothesis is correct, different parts of the Vh locus will be adjacent to DJ in different pro-B cells, and those regions will now also have H3K4me3, attracting Rag2. We will therefore determine the transcriptional profile of individual pro-B cells using high-throughput microfluidic Fluidigm technology, and the same single cell cDNA will be sequenced to determine the VDJ rearrangement. We hypothesize that only a subset of germline transcripts is expressed in each cell and that there will be a correlation of rearrangement of individual Vh genes with a particular transcriptional profile. Having a diverse repertoire of antibodies is critical to be able to combata wide variety of pathogens. This novel hypothesis will change the paradigm of how a diverse repertoire of antibodies is created.

PUBLIC HEALTH RELEVANCE: The knowledge that we gain here will shed light on the long standing question of how non-coding germline transcription influences accessibility for V(D)J rearrangement, and also will shed light on the transcriptional heterogeneity as pro-B cells differentiate and the role that this heterogeneity plays in influencing V(D)J recombination and the generation of a diverse antibody antibody repertoire which is essential to combat a wide variety of pathogens. Misregulation of V(D)J rearrangement can result in translocations resulting in lymphomas and leukemias, and recent data have supported the hypothesis that the 3D structure of the locus, and proximity of transcribed genes in transcription factories, can predispose genes such as c-Myc to translocate to Igh. Thus, the information that we gain in this proposal will aid in our understanding of the etiology of these diseases, as well as in the production of altered antibody repertoires in autoimmunity and immunodeficiency.