Transcriptional heterogeneity within pro-B cells

原B细胞内的转录异质性

• 批准号: 8569969
• 负责人: ANN J FEENEY
• 金额: $ 26.72万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-16 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8569969
• 关键词: Antibodies; Antibody Repertoire; Autoimmunity; B cell differentiation; B-Cell Development; B-Lymphocyte Subsets; B-Lymphocytes; Biological Assay; Cells; Complementary DNA; Cytokine Receptor Gene; DNA Sequence Rearrangement; Data; Development; Disease; Ectopic Expression; Epigenetic Process; Etiology; Functional RNA; Gene Expression Profile; Gene Rearrangement; Generations; Genes; Genetic Recombination; Genetic Transcription; Heterogeneity; Histones; Immunologic Deficiency Syndromes; Individual; Knowledge; Light; Messenger RNA; Microfluidics; Molecular Profiling; Nuclear Structure; Pattern; Play; Production; Proteins; RNA; Rag1 Mouse; Recruitment Activity; Role; Sampling; Signal Transduction; Sorting - Cell Movement; Staging; Susceptibility Gene; System; TCF3 gene; TF gene; Technology; Transcript; Ursidae Family; V(D)J Recombination; base; c-myc Genes; combat; differentiated B cell; insight; leukemia/lymphoma; novel; pathogen; programs; promoter; public health relevance; three dimensional structure; transcription factor; transcriptome sequencing

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.

描述（由适用提供）：细胞分化由转录因子指导，众所周知，转录因子和其他蛋白质的mRNA水平随着细胞的分化而变化。然而，通过单细胞表达分析的最新发现表明，在单个细胞的转录谱中，即使是相同表型发育阶段的转录谱的异质性。尚未进行此类研究来确定单个pro-B细胞之间的潜在转录异质性，并确定它随着细胞在pro-B细胞发育过程中的差异步骤而变化。重要的是，在Pro-B细胞阶段，IGH V（D）J重组发生，我们做出了一个新的假设，即转录异质性对于生产多样性抗体库的生产至关重要。已经提出了通过V基因的非编码种系转录，以使可访问重组的基因。种系转录的一个作用是用表观遗传标记H3K4me3标记抄录区域，该区域可以直接募集RAG2。我们已经表明，非编码种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（以及某些V基因）与E e胚系转录本的启动子E e e近距离接近。 e？oos是从DJ重排的1-2 KB，一个V基因将重新排列。这是我们假设种系转录直接导致IGH基因座压实，我们为我们通过对IGH转录组的RNA-SEQ分析确定的两个主要反义种系转录启动子证明了这一点。但是，如果只有在这些主要种系笔录中转录的区域位于DJ重排附近，则可以预测这些区域附近的V基因比其他V基因更有可能重新排列，但事实并非如此。 IGH基因座VH部分中其他种系转录本的水平通常很低。尽管通常认为大多数功能性VH基因在Al Pro-B细胞中以低水平转录，但我们提出了不同的假设。基于单个细胞转录分析的新兴数据，我们提出，pro-B细胞之间存在转录异质性，以使每个pro-b细胞表达不同水平的关键转录因子或可能的随机性的可能影响的种系转录本的不同子集。如果该假设正确，则VH基因座的不同部分将与不同的Pro-B细胞中的DJ相邻，现在这些区域也将具有H3K4ME3，吸引RAG2。因此，我们将使用高通量微流体流体技术来确定单个Pro-B细胞的转录谱，并将对相同的单细胞cDNA进行测序以确定VDJ重排。我们假设在每个细胞中只表达了种系转录本的子集，并且单个VH基因的重排与特定的重排相关 转录轮廓。拥有抗体的潜水员曲目对于能够对抗各种病原体至关重要。这个新颖的假设将改变如何创建潜水员曲目的范式。