The hematopoietic microRNome can be set apart from that of somatic cells and tissues by a distinctive miRNA signature, differential expression patterns of let7, and a unique 5p:3p distribution for selected miRNAs. Previous studies uncovered miRNAs circumscribed to or enriched in the immune system, such as miR-150, miR-155, miR-223, miR-146, and the miR-181 family (Barski et al., 2009
; Basso et al., 2009
; Chen et al., 2004
; Landgraf et al., 2007
; Merkerova et al., 2008
; Monticelli et al., 2005
; Neilson et al., 2007
; Taganov et al., 2006
; Wu et al., 2007
; Zhang et al., 2009
). Our comparative cluster analysis corroborated these findings and further assigned previously characterized miRNAs to the hematopoietic system: miR-15b (nTreg), miR-26a/b (neutrophils), miR-28 (GC B cells), miR-107 (basophils), miR-320 and miR-148a (plasma cells), miR-128 (CD4+
), miR-221 and miR-222 (Tfh-basophils), miR-147 (Th1), miR-182 (NK cells), and miR-193b (HPCs). Of note, both miR-28 and miR-148a were also highly specific for human GC and plasma cells, respectively (Figure S6). In addition, we identified 14 mouse miRNAs that show preferential expression in various cells of the immune system In the context of gene discovery, it is important to point out that of the >300,000,000 sequence reads analyzed, as many as 2,406 putative mouse miRNAs were predicted based on a probabilistic model of miRNA biogenesis ((Friedlander et al., 2008
), not shown). Of these, however, only a small fraction (18 in total) was detected at amounts greater than 100 TPM. It is interesting to note therefore that the overwhelming majority of putative miRNAs uncovered by our analysis appear to represent low abundant hairpin species that might on occasion be processed by the RNAi pathway. Admittedly, we cannot rule out that some of these putative miRNAs are expressed at considerable levels in tissues not examined in our survey. Furthermore, the arbitrary 100 TPM threshold is not a predictor of physiological activity, which in principle could still be significant at very low levels of miRNA expression. In light of our findings however, it is reasonable to propose that most mouse and human miRNAs have already been characterized. This idea is supported by the fact that recent large-scale cloning efforts yielded few novel, mostly low abundant, miRNAs (Basso et al., 2009
; Landgraf et al., 2007
The combining high-throughput miRNA-seq, mRNA-seq, and ChIP-seq provides an unprecedented view of the various regulatory steps that shape miRNA expression and abundance during lymphopoiesis. At the epigenetic level, the methylation status of H3K27 partitioned lymphocyte-specific miRNA genes into two distinct subsets. Those belonging to the miR-139/miR-147 group were found to retain the repressive H3K27me3 mark throughout development up until miRNA gene transcription was elicited. miRNA genes of the miR-155/145561_chr11 group were H3K27 demethylated at earlier stages of development, and thus they appeared to be poised for activation prior to full transcription. The rationale for this subdivision might be provided by the spatiotemporal context in which these miRNAs are expressed. miR-147 for instance is induced as naïve CD4+
T cells differentiate into the Th1 lineage. This process of fate determination relies on positive epigenetic reprogramming and expression of key factors, and negative regulation of competing pathways driving differentiation to other T helper cell types (Wei et al., 2009
). Similarly, miR-139 derepression occurs as part of another fate determination step, from pluripotent hematopoietic progenitor cells to pro-B cells. Although the precise role of miR-139 and miR-147 remains to be determined, the expectation would be that miRNAs involved in symmetric or progressive lymphocyte lineage commitment would be tightly regulated by robust mechanisms. One such mechanism may be polycomb group-mediated H3K27 trimethylation, which might ensure induction of cell fate determination only at the appropriate time.
Our data have also revealed lymphocyte-induced miRNA genes that appear to be epigenetically poised for transcription early in development. These miRNA genes are associated with some activating histone marks, are H3K27me3 depleted, recruit low levels of PolII, but lack H3K36me3 or H3K79me2 –hallmarks of polymerase elongation. This sub-induced chromatin state is reflected by the low, but detectable, miRNA expression prior to full gene transcription. For miR-155, 145561_chr11, and other miRNAs under this category, full expression occurs as resting lymphocytes are exposed to mitogens and cytokines typically released during the immune response. The benefits of maintaining critical miRNA genes like miR-155 in a preexisting sub-induced state maybe best understood in the cadre of viral or bacterial infections, which require rapid T and B cell responses. Based on these considerations, it will be important to determine whether miRNAs strictly dependent on H3K27me3 for expression are involved in lymphocyte developmental decisions, while H3K27me3 independent ones drive B and T cell activation. Ongoing studies indicate that this might be indeed the case.
Transcriptionally, we have shown that expression of at least one quarter of mature miRNAs closely follows spliced primary transcripts as determined by mRNA-Seq. Because mRNAs are stabilized by polyadenylation and other mechanisms, this is likely to be an underestimate, e.g. the correlation between mature miRNAs and unspliced
pri-miRNAs is expected to be more significant. Regardless of the precise number, the results were nonetheless unexpected in light of the fact that studies with cell lines fail to find any such correlation (Thomson et al., 2006
). Transformed cells, however, display a global reduction in mature miRNAs (Lee et al., 2008
; Lu et al., 2005
), presumably from a general deficiency in miRNA processing. Alternatively, in primary cells several post-transcriptional mechanisms are bound to promote deviations between transcription rate and mature miRNA expression, such as efficiency of miRNA processing, miRNA editing, or miRNA nuclear transport. In addition to these, we have found that ectopically expressed mRNA targets can both increase or decrease a particular miRNA pool. Whether endogenous mRNAs can likewise influence fluctuations in miRNA abundance remains to be determined. Nevertheless, it is reasonable to propose that an increase in the absolute number of mRNA targets may impact the balance and/or half-life of “free” miRNA, miRNA-RISC, and miRNA-RISC-mRNA complexes. We find this scenario intriguing, as it raises the possibility that under physiological conditions, miRNAs and mRNAs regulate each other’s homeostasis.
In summary, our studies reveal some of the epigenetic, transcriptional, and posttranscriptional strategies that help orchestrate cellular abundance of miRNAs during lymphopoiesis. The hematopoietic miRNA signatures provided by the data represent a valuable resource that will help guide future gene-targeting experiments of individual miRNAs. In this respect, a major challenge in the field has been the identification of critical miRNAs:miRNA targets driving developmental decisions. A strategy to solve this problem is based on the observation that cellular concentration of miRNA targets fluctuates as a function of cognate miRNA expression (e.g. miR-150:c-Myb, (Xiao et al., 2007
)). In principle, by applying microsequencing and bioinformatics to a large number of developmental stages it should be possible to predict functional miRNA:miRNA targetpairs. Our preliminary studies using the miRNA- and mRNA-Seq data from B cell development support this view. We anticipate that genomic approaches such as this will help unravel how miRNAs regulate development and effector functions of the immune system.