The initiator factor of hypermutation and recombination is AID, a B cell specific enzyme that is only expressed in B lymphocytes directly involved in the immune response. Because of AIDs link to tumor development, B cells have developed a variety of mechanisms that curtail its activity in vivo. As we have recently showed in collaboration with Dr. Papavasiliou from the Rockefeller University, one prominent mechanism by which AID is regulated is via microRNA activity. microRNAs (miRNAs) are non-coding small RNAs that modulate the cells proteome by annealing to 3-unstranslated regions of cognate mRNAs and inhibiting protein translation and/or promoting mRNA instability. Since their discovery in C. elegans 16 years ago, miRNA orthologs and paralogs have been described in a variety of species, suggesting these regulatory RNAs are involved in basic cellular functions across the phyla. In the mammalian genome, microRNAs are encoded within introns of protein-coding genes or as independent entities transcribed either by RNA polymerase II or RNA polymerase III. In some instances, groups of miRNAs are also clustered and processed from a single transcript. Due to the their palindromic nature, miRNAs in nascent primary transcripts (pri-miRNAs) display a characteristic stem-loop structure that in the nucleus is recognized and cleaved by the Drosha/DGCR8 complex into 60-70 nt precursor (pre) miRNAs. Once in the cytoplasm, pre-miRNAs are further processed by the RNase III endonuclease DICER into mature RNA fragments of 22 nt in length, which are loaded into the RNA-silencing complex RISC. Partial sequence complementary between the 5 end of the mature miRNA (6-8 nt seed region) and its target mRNA leads to downregulation of protein expression. As is the case for non-hematopoietic tissues, lymphocytes and other cells of the immune system rely on miRNAs to effect lineage commitment, proliferation, migration, and differentiation. In most cases, these activities are orchestrated by both ubiquitously expressed and hematopoietic specific miRNA species, which upon deletion or overexpression impair the immune system at various levels. In like manner, conditional ablation of DICER or other miRNA processing factors results in a profound block of both B and T cell development. In apparent contrast to these striking phenotypes, it is notable that miRNAs control hematopoiesis through small changes in the cellular concentration of key factors. In the B cell compartment for instance, mir150 curtails c-Myb activity in a dose-dependent fashion over a narrow range of miRNA and c-Myb concentrations. Similarly, mass action seems to be the underlying principle behind mir155 regulation of AID, or mir17-92-mediated inhibition of PTEN and Bim proteins. In retrospect, the graded miRNA activity shown in these examples provides a rationale to the happloinsufficiency previously observed in AID, cMyb, PTEN, and Bim heterozygous mice. This basic principle however is not restricted to the mammalian immune system as data from a wide variety of experimental systems increasingly demonstrate that the absolute concentration of miRNAs is crucial at managing a cells proteome. Yet, how miRNA levels are controlled upon differentiation of specific cell lineages remains to be explored. One major limitation in measuring miRNA abundance during ontogeny has been the lack of quantitative, systematic approaches to profile small RNAs with high accuracy. In a manuscript in preparetion, we have used massive parallel sequencing to monitor miRNA changes in a large number of B and T cell developmental stages. To define lymphocyte-specific miRNA signatures, the microRNome of other hematopoietic lineages and samples representing diverse mouse cells and tissues was also characterized. Furthermore, by means of genome-wide chromatin immunoprecipitation (ChIP-seq) and high-throughput mRNA sequencing (RNA-seq), we have assessed the epigenetic status of miRNA genes concomitant with pri-miRNA expression during lymphopoiesis. This approach provided a comprehensive view of the epigenetic, transcriptional, and indirectly, posttranscriptional mechanisms shaping miRNA cellular concentration. The data partitioned miRNAs into distinct subsets. Epigenetically, the great majority of lymphocyte-specific miRNAs, which are silenced outside the immune system, were found to be H3K27 demethylated in hematopoietic stem cells (HSCs). Demethylation however did not necessarily promote pri-miRNA gene transcription, which in most cases was only fully induced in subsequent T or B cell developmental stages along with dramatic increases in acetylating of histone H3K36 and H3K14, and methylation of H3K36 and H3K79. In contrast to this large group, expression of a small minority of miRNAs were more tightly regulated as they maintained H3K27me3 up until full gene transcription was induced. In the context of gene expression per se, our data shows that up to one quarter of mature miRNAs mirror the relative expression profiles of pri-miRNA transcripts during differentiation. Finally, we provide evidence suggesting deviations in pri-miRNA/miRNA expression profiles can be attributed at least in part to target mRNA levels, which seem to directly influence cellular concentration of cognate miRNAs. Altogether, our findings shed light on the mechanisms that fine-tune expression and cellular abundance of miRNAs during lymphocyte differentiation. The quantitative microRNome profiles from a large number of mouse cells and tissues, the discovery of 19 novel, phylogenetically conserved miRNAs, and the systematic classification of miRNAs based on their epigenetic and transcriptional regulation constitute an invaluable resource for a wide range of future miRNA studies.
