Transcriptional heterogeneity within pro-B cells

描述(申请人提供)：细胞分化是由转录因子引导的，众所周知，转录因子和其他蛋白质的mRNAs水平随着细胞分化而变化。然而，单细胞表达分析的最新发现揭示了单个细胞转录谱的不同质性，即使是相同表型发育阶段的细胞也是如此。还没有这样的研究来确定单个Pro-B细胞之间潜在的转录异质性，以及在Pro-B细胞发育过程中，当细胞经过分化步骤时，它是如何变化的。重要的是，在原B细胞阶段，IgH V(D)J重组发生，我们提出了新的假设，即转录异质性对于产生不同的抗体库至关重要。通过V基因的非编码生殖系转录已经被提出，以使基因可用于重组。生殖系转录的一个作用是用表观遗传标记H3K4me3标记转录区域，H3K4me3可以直接招募RAG2。我们已经证明，非编码生殖系转录的另一个关键作用是改变IgH基因座的3D结构，使转录区域(从而使一些V基因)更接近I？生殖系转录本的启动子E？E？距离DJ重排1-2kb，其中一个V基因将重排到该重排。因此，我们假设生殖系转录直接导致免疫球蛋白基因座紧凑，并且我们通过对免疫球蛋白转录组的RNA-SEQ分析确定了两个主要的反义生殖系转录启动子。然而，如果只有在这些主要生殖系转录本中转录的区域位于DJ重排附近，这将预测这些区域附近的V基因比其他V基因更有可能重排，但情况并非如此。在IgH基因座的VH部分，其他生殖系转录本的水平通常较低。虽然人们普遍认为大多数VH功能基因在a1-Pro-B细胞中低水平转录，但我们提出了不同的假设。基于单细胞转录分析中出现的数据，我们认为在Pro-B细胞之间存在转录异质性，使得每个Pro-B细胞表达不同的生殖系转录子集，可能受关键转录因子水平的差异影响，也可能是随机的。如果这一假设是正确的，VH基因的不同部分将在不同的PRO-B细胞中与DJ相邻，这些区域现在也将有H3K4me3，吸引Rag2。因此，我们将使用高通量微流控技术来确定单个Pro-B细胞的转录图谱，并对相同的单细胞cDNA进行测序以确定VDJ重排。我们假设在每个细胞中只表达生殖系转录子的一个子集，并且单个VH基因的重排与特定的 转录档案。拥有不同的抗体库对于能够对抗各种各样的病原体至关重要。这一新的假说将改变一系列抗体如何被创造出来的范式。

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.