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Immunology. 2008 May; 124(1): 23–32. doi: 10.1111/j.1365-2567.2008.02818.x. | PMCID: PMC2434389 |
Copyright © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd Genomics and the immune system Matthew E Pipkin1 and Silvia Monticelli2 1Immune Disease Institute and Harvard Medical School, Boston, MA, USA 2Institute for Research in Biomedicine, Bellinzona, Switzerland Received November 19, 2007; Revised January 22, 2008; Accepted January 23, 2008. While the hereditary information encoded in the Watson–Crick base pairing of genomes is largely static within a given individual, access to this information is controlled by dynamic mechanisms. The human genome is pervasively transcribed, but the roles played by the majority of the non-protein-coding genome sequences are still largely unknown. In this review we focus on insights to gene transcriptional regulation by placing special emphasis on genome-wide approaches, and on how non-coding RNAs, which derive from global transcription of the genome, in turn control gene expression. We review recent progress in the field with highlights on the immune system. Keywords: epigenetic, genome-wide, lymphocytes, non-coding RNAs, transcription The continuous development of naïve lymphocytes and their activation during pathological and homeostatic processes provides an unparalleled set of differentiation and activation programmes to examine and manipulate in order to understand genome biology. Essentially complete versions of human and mouse genome sequences are now available. However, knowing the sequence is only part of the story. Because eukaryotic genomes are packaged into chromatin, depending on the particular cellular context, only a subset of genomic sequence is available to the enzymatic processes that extract the information contained in the underlying sequence. Patterns of chromatin accessibility determine expression, recombination, repair and replication of the genome and, like the sequence itself, can be inherited ‘epigenetically’ through successive cellular generations by chemical modifications to the proteins and DNA that comprise chromatin. 1,2 In addition, chromosomes in the interphase nucleus are organized into a looped, three-dimensional architecture. These configurations are dynamic and facilitate regulated spatial interactions between distal chromosomal regions, and proximity of genes to subnuclear domains enriched with the enzymatic complexes that manipulate the DNA sequence. 3,4Unexpectedly, the majority of the genome is transcribed, resulting not only in protein coding mRNAs, but also a large number of non-coding RNAs (ncRNAs). 5 Some ncRNAs have known functions in regulating expression of coding mRNAs at a transcriptional or post-transcriptional level. 6 Surprisingly, the cadre of protein coding transcripts that are expressed by different immune cell-types are not dramatically different, whereas the larger disparity exists in the levels of their expression. 7 Therefore, quantitative differences in the transcript levels of coding genes appears to be an important mechanism that underpins immune cell diversity, which can be shaped by differences in the expression of ncRNA; in many cases this might be determined post-transcriptionally. Taken together, genome sequence and its protein-coding segments are only the tip of the iceberg. Emerging studies of how epigenetic, three-dimensional and non-coding RNA mediated regulation of the genome are integrated to generate diversity and to govern responsiveness of the immune system indicate that the central dogma of molecular biology is more sophisticated than once perceived. In this review we focus on some of the first genome-wide studies of chromatin to highlight the potential for developing global perspectives of the how accessibility to genome sequence is configured, and also discuss recent experiments that examine the role of intergenic transcription and non-coding RNA mediated control of gene expression. Nucleosome positioning genome-wide and locations of chromatin accessibility Nucleosomes are the fundamental repeating subunits of chromatin. They facilitate multiple layers of genome condensation, but also mediate its access. A single nucleosome consists of an octamer of histone proteins, the globular regions of which form a core particle around which 147 bp of DNA is wrapped. 8 Only a short linker DNA region of variable length exists between adjacent nucleosomes, such that up to 90% of the genome may be packaged into nucleosomes and inaccessible to other regulatory factors. 9 Using tiled microarrays, nucleosome positions have now been mapped genome-wide in yeast, and across the promoters of 3692 genes in multiple human cell-lines, which has illuminated several global features that are depicted in . 9–11 Perhaps one of the most compelling concepts revealed by these studies is that the genomic DNA sequence itself encodes an intrinsic programme for preferred nucleosome positions. 9 Up to 50% of nucleosome positions could be predicted based on sequence alone; the remainder are likely to be determined by the lineage-restricted subset of sequence specific DNA binding proteins (conventional transcription factors) expressed in a given cell-type, which collaborate with chromatin remodelling enzymes that can reposition nucleosomes. 8,12Transcription factor binding regulates nucleosome positions and this alters the finite accessibility profile of the genome and yields at least two outcomes: to poise certain subsets of the genome for differential transcriptional outputs (differentiation), or to acutely modify transcription of previously poised genes (activation/repression). How do cells reprogram their genomes to make appropriate lineage choices? The differentiation of CD4 T cells into T helper 1 (Th1) cells provides a good example for how transcription factors can coordinate nucleosome remodelling and lineage choices. Naïve CD4 T cells require antigen stimulation and specific cytokine signals to differentiate into Th1 effector cells, which selectively transcribe the Ifng gene. 13 This involves activation of both Th1 lineage-specific and widely expressed transcription factors (T-bet, and nuclear factor of activated T cells (NFAT), respectively) and chromatin remodelling. 14,15 In naïve T cells, a positioned nucleosome exists over known T-bet binding elements immediately upstream of the transcriptional start site (TSS). However, quickly after T-cell receptor (TCR) stimulation under conditions that promote Th1 cell development, the bramah-related gene 1 (Brg-1), an enzymatic component of the switch (Swi)-sucrose non-fermenter (SNF) chromatin remodelling complex is recruited to this region and nuclease accessibility develops at the TSS indicating nucleosome re-positioning. One potential explanation for how these factors collectively remodel the Ifng promoter leading to transactivation could be the intrinsic placement of NFAT binding sites in the promoter. A conserved NFAT element is located in the accessible linker region (i.e. inherently nucleosome-free in T cells) upstream of the positioned nucleosome at the Ifng TSS. NFAT is activated quickly upon TCR stimulation and appears to initiate Brg-1 mediated nucleosome remodelling at the TSS; that is, the phosphatase activity that drives NFAT nuclear translocation is required to recruit Brg-1 to the Ifng TSS, and for nuclease sensitivity. This likely provided a moment of entry for T-bet, which is not expressed in naïve cells, but is quickly induced by TCR signalling, to access its normally obscured binding sites. Thus, accessible binding sites for a widely expressed transcription factor (NFAT) can initiate access to lineage specific binding sites for specific factors (T-bet) that are induced during differentiation, and in turn result in stabilized re-positioning of nucleosomes yielding a gene that is poised for subsequent high-level lineage restricted transcriptional activation. Specifying transcriptional responses with sequence specific DNA binding factors Conventional transcription factors operate combinatorially and co-operatively at cis-regulatory sequences to govern chromatin accessibility and transcription. Consequently, the role of any individual transcription factor is exquisitely dependent on the context in which it is engaged. For example, NFAT1 is recognized well for its capacity to induce interleukin-2 (IL-2) transcription in cooperation with AP-1 in activated effector CD4 T cells, in part by binding to a composite sequence motif of the IL-2 promoter termed the antigen receptor response element (ARRE). 16 However, NFAT can partner with forkhead box protein 3 (Foxp3) in regulatory CD4 T cells (Tregs) and negatively regulate IL-2 transcription. 17 Foxp3 is selectively expressed in Tregs, is required for Treg function and is sufficient for many of these, such as IL-2 repression, when expressed ectopically in conventional CD4 T cells. 17,18 Crystal structure of the DNA binding domains (DBDs) of NFAT1 and that of Foxp2 (a similar FOXP family member) bound in complex with ARRE2 DNA indicates that the FOXP DBD is situated on the DNA similarly to AP-1 in the conventional NFAT1/AP-1 complex. 17It is unclear mechanistically how NFAT–Foxp3 interactions negatively regulate the transcription of IL-2. From analysis of the crystal structure, one hypothesis is that Foxp3 competes with AP-1 for DNA binding; a consensus FOXP binding sequence exists within the AP-1 binding motif. 17 Alternatively, the NFAT–Foxp3 interaction might also function by recruiting co-repressor complexes that actively repress transcription; Foxp3 directly associates with repressive complexes in vivo.19 Structure-guided Foxp3 mutants that are predicted to disrupt its interactions with NFAT1 are less efficient at repressing expression of IL-2 when expressed in primary CD4 T cells. 17 These results suggest that the NFAT1–FOXP3 interface may be important for the ability of Foxp3 to bind at the IL-2 promoter, and either compete for NFAT/AP-1 mediated transcription or actively repress the IL-2 promoter. Transcription factor binding on the ARRE2 element of the IL-2 promoter is cooperative and depends on direct protein–protein interactions with adjacent factors. 16,20 However, this type of co-operativity might only be one variation on a theme of combinatorial transcription factor binding at cis-regulatory domains. Assembly of transcription factors on the interferon-β (IFN-β) enhanceosome is also co-operative, but it does not rely on extensive protein contacts between adjacent factors. Rather, co-operative binding is mediated by local changes in DNA conformation, which results from adjacent factors binding on overlapping consensus binding-motifs. 20,21 Precise sequence organization of series of overlapping elements results in the factors contacting all 57 bp of the enhanceosome, which strictly specifies the appropriate assembly of a defined combination of seven transcription factors and creates a continuous protein interface upon the enhanceosome surface. 20 A central conclusion is that regulation of the enhanceosome via its entire sequence as a unit, rather than regulation of its individual elements, is the basis for its function in IFN-β gene regulation. The cooperative binding of factors on the IFN-β enhanceosome confers an enormous degree of specificity to the IFN-β transcriptional response, allowing it to only respond upon integration of the appropriate extrinsic signals, but is unlikely to account for the strong synergy characteristic of its regulation in vivo.20 One possibility to account for this disparity is that the synergy resides at the level of different co-activators that are recruited to the assembled enhanceosome. Multivalent interactions with co-activators are important for activating transcription by the IFN-β enhanceosome. 22 Therefore, the specific arrangement of the transcription factors at the assembled nucleosome is likely to provide the most efficient docking site for co-activators responsible for recruiting and stimulating processivity of the RNA Pol II machinery. In theory, these functions could be mediated through long-range interactions with additional cis-regulatory regions in the locus, an idea that extends the premise of co-operativity from individual elements, to full enhanceosomes, to the entire transcriptional regulation of a given locus. Zeroing in on long-range cis-acting domains A major thrust in genome regulation is to identify the genome-wide array of cis-acting domains that are controlled by transcription factors. Within the immune system, it is known that multiple proximal and distal DNA cis-regulatory domains, such as promoters, enhancers, silencers, locus control regions (LCRs) and insulators coordinate processes such as somatic recombination of immunoreceptor genes and transcription. 2 Active cis-regulatory domains are often, if not always, associated with a remodelled chromatin structure that renders the underlying DNA, by comparison to surrounding chromatin, exquisitely sensitive to cleavage with nucleases such as DNase I. This has now been exploited to identify active cell-type specific cis-regulatory regions. Two general approaches, either using a chip array or a sequencing based strategy, have now been applied toward identification of DNase I hypersensitive sites (DHSs) genome-wide. 23–27 These approaches provide promise that determining the location of all active regulatory domains in given cellular contexts is becoming a reality. In the largest sequencing-based approach to date, a total of ~15 000 DHSs were identified in resting human CD4 T cells (). 25 Perhaps surprisingly, only 10% of DHSs were lymphoid specific; the rest were common between lymphocytes, hepatocytes and endothelial cells. Moreover, no significant differences were found between CD4 and CD8 T cells, suggesting that these cell types are very similar. This could reflect the low number of genes that appear to be differentially expressed in different lineages in the immune system. 7 However, it might also emphasize current technological constraints of genome-wide DHS mapping approaches, because traditional methods to detect DHSs have clearly established that a number of DHSs form differentially in CD4 versus CD8 T cells, for example, in the CD4 locus. 28 The lack of sensitivity in the sequencing based approach likely relates to the number of clones that must be sequenced to identify all DHSs, which could be orders of magnitude higher than those analysed to date. 25 Although microarray-based approaches could provide better sensitivity, 27 the capacity to analyse even larger data-sets will be required to identify all DHSs genome-wide, especially in the context of closely related cell-types. While discerning global networks of active regulatory regions using genome-wide approaches will be essential, the dimensions of individual genomic regions that encompass most or all regulatory regions essential for the specific and synergistic control of a given locus might not be that immense. Several immune system related loci such as that of the major histocompatibility complex (MHC), Ig and TCR genes are extremely large (Megabases), but many more are unlikely to exceed a few hundred kilobases. The Cd4, Cd8, Ifng, Il-4/13/5 (Th2 cytokine) and Prf1 genes, all appear to be contained in loci <200 kb. 29–31 Therefore, focused studies of individual loci at high resolution are likely to remain critical. A long-range method has been developed that can identify traditional DHSs at ranges of 100 kb. 32 This simple approach facilitates determining most or all cis-acting regions for even a large locus in the context of a single study. Deciphering the formation of cell-type specific patterns of DHSs can be used to infer their potential function, although it still remains only a measure of localized chromatin accessibility, and even more detailed approaches are critical for implying the specific function of these domains. Some of this information is found in the signature of post-translational modifications to chromatin that occur over these regions. Histone modification profiles of annotated features genome-wide The amino terminal ‘tails’ of the histone proteins of nucleosomes are unstructured; they protrude from the core particle and are not directly involved in wrapping DNA into nucleosomes. 8 However, histone tails participate in intramolecular contacts within the chromatin fiber to promote higher order chromatin folding, and are also the scaffold upon which chromatin modifying complexes are recruited to alter chromatin structure and remodel nucleosome positions. Up to eight classes of chromatin modifications targeted to at least 60 distinct residues in nucleosomal histones are now known and their specific roles are beginning to emerge. 8 Initial genome-wide studies in T lymphocytes are now laying the foundation for deciphering a global logic behind regulatory configurations of chromatin structure. 33,34In resting human CD4 T cells, the genome-wide profile of diacetylated histone H3 at lysines 9 and 14 (H3K9 and H3K14), 34 and the distributions of 20 histone lysine and arginine methylations, one histone variant (H2AZ), RNA polymerase II (Pol II), and the insulator regulatory factor CCCTC-binding factor (CTCF) have been mapped. 35 These studies reveal the first comprehensive cross-section of how chromatin is configured at annotated genomic features (). Extended regions of accessible chromatin defined by nuclease sensitivity and histone acetylation, ‘active chromatin domains’, were once perceived to encompass broad regions surrounding developmentally appropriate genes. 36 However, genome-wide studies now suggest that they are actually formed by punctate regions of accessible chromatin characterized by ‘islands’ of histone modifications. 34,35 These islands primarily occur at TSSs, CpG islands, DHSs and conserved non-coding sequences (CNSs), although a notable number do not co-localize with any currently annotated feature, but nevertheless identify sequences with regulatory function. 37 On a more focused level, epigenetic profiling of the Ifng locus in naïve and activated CD4 T cells is consistent with these genome-wide observations and also provides a detailed model for the regulated changes that occur to the locus during helper T-cell differentiation. 31,38In contrast to the punctate type patterns of histone modifications found in flanking intergenic sequences, the transcribed aspect of genes exhibit a more uniform distribution of histone modifications. 35 Active transcription units form domains characterized by a number of ‘activating’ histone modifications, whereas inactive transcription units form distinct domains consisting of modifications characteristic of silent chromatin (). The chromatin modification signature of active genes and TSSs turns out to be highly predictive of unannotated transcriptional units. 35 Thus, the chromatin modification profile of different genomic features is likely to become an important discovery tool to complement current approaches to computational sequence analyses. Moreover, the chromatin signature of genes is likely to ultimately be a better indicator of the developmental potential of cells because chromatin analyses can distinguish genes that are silent, from those that are poised for transcription, or actively transcribed. Therefore epigenetic, rather than only transcript, profiling of coding genes during immune cell development will most likely provide important insights as to how cells settle on lineage decisions. 39Long-range genome organization in the nucleus and regulation of gene activity The regulation of chromatin structure and genome activity appears to be intimately associated with specific subnuclear compartments. In the interphase nucleus, chromosomes adopt preferred, non-random positions in relation to the nucleus itself and to neighboring chromosomes. Chromosome territories are defined cytogenetically as the distinct subdomains of the nucleus in which interphase chromosomes occupy. Within chromosome territories, mammalian chromatin appears to be organized into megabase sized loops that are linked by common anchor points. 4 Within these loops, regulatory processes such as replication appear to be coordinated and inherited. It is now clear that repositioning of genes from chromosome territories is linked to gene function, perhaps by delivering them into different subnuclear regions that are enriched for specific trans regulatory factors. Thus, analogous to repositioning of nucleosomes along the DNA strand, genome access is also governed by reprogramming the looped architecture of chromosomes. The MHC locus is governed by long-range repositioning of chromosome territories during gene-activation. IFN-γ signalling associated with inflammation triggers transcriptional activation of genes in the class II MHC locus on human chromosome 6. IFN-γ stimulation quickly drives the extrusion of the class II locus beyond the perimeter of the resting chromosome 6 territory and is coordinated with the ordered binding of transcription factors and chromatin remodelling enzymes to multiple promoters in the locus, leading to induction. 40 One potential concept supported by these observations is that genes induced during development and activation must be repositioned to regions in the nucleus that are enriched in regulatory factors that promote functions such as transcription or recombination. Indeed, RNA Pol II appears to be enriched in dense foci in the nucleus and to associate with activated genes, small subdomains that have been termed transcription factories. 3 In addition, developmental activation of high-level β-globin gene transcription during erythropoiesis is coordinated with repositioning of the locus into a transcription factory, which requires its distal LCR. 41 Thus, nuclear repositioning and association with distinct nuclear subdomains is necessary for normal transcription, and appears to be mediated through distal cis-acting sequences. Related to global repositioning of loci in the nucleus, communication between individual cis-acting regions in genes appears to be a critical event during both transcription and immunoreceptor recombination. The chromosome conformation capture (3C) assay and its derivatives provide a biochemical strategy to assay the spatial and physical proximity of distal cis-acting sequences. 42–44 Their application to the Th2 cytokine locus has provided interesting insights regarding how multiple cis-acting regions throughout the locus efface during CD4 T-cell differentiation and activation. 45 Cai et al. have now also provided evidence that the genome organizer special AT-rich sequence binding 1 (SATB1) anchors the looped topology of the Th2 cytokine locus and supports its remodelling during transcriptional induction. 42 In the locus, SATB1 bound nine predicted elements as well as two CNS regions, in a differentiated Th2 cell-line under resting conditions. In this context, the locus is organized into at least two large loops by SATB1 binding sequences located its 5′ and 3′ termini. Upon activation, multiple internal contacts develop which form tight loops, and that bring the promoters of Il-4 and Il-13 into proximity with distal elements in the LCR and CNS-1; the base of these loops was fastened by SATB1. Bound SATB1 co-localized at the locus with multiple trans factors known to regulate the Th2 cytokine locus, and is also known to interact directly with chromatin remodelling proteins. 2 SATB1 and its homologue SATB2 have tissue and activation specific expression patterns, thus they are both likely to be key players in anchoring functional loop domains within chromosome territories to control proximity of distal cis-acting regions that control genome function, in the immune system and beyond. High-throughput genome analyses and genome-wide transcription As detailed above, multiple genome-wide approaches have now documented that a substantial fraction of sequences located in distal locations relative to protein-coding genes are likely to be functional. Coincident with these findings, recent work also indicates much more of the genome is transcribed than what was once thought. Large-scale studies of the human and mouse genomes have revealed that although there are more than 21 000 protein-coding genes, significantly larger portions of both genomes are transcribed (69 185 gene predictions in human and 71 259 in mouse), and gene number does not scale with complexity of an organism as previously thought. In addition to protein-coding mRNAs and housekeeping RNAs (like rRNAs and tRNAs), different kinds of regulatory ncRNAs are constantly being discovered. Identified functional ncRNAs range in size from ~22 nt of microRNAs (miRNAs) to ~2·2 kb of HOTAIR (HOX antisense intergenic RNA), ~18 kb of XIST (X-inactive-specific transcript) and ~108 kb of AIR (antisense IGF2R RNA) ncRNAs. 46–48 Estimates made by the Encyclopedia of DNA Elements (ENCODE) consortium suggest that the human genome is so extensively transcribed that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that overlap one another. 5 Therefore, the process of intergenic transcription, and the resulting ncRNAs that are generated, could each provide a critical link between long-range control of genome accessibility and function (). Non-coding RNAs: transcriptional and post-transcriptional gene silencing Non-protein coding transcripts, as well as the process of transcription per se can have a role in regulating gene expression both at a transcriptional and post-transcriptional level. The yeast Schizosaccaromyces pombe provides an example of short ncRNAs that are important for epigenetic modifications and transcriptional gene silencing (TGS), with a mechanism that involves the RNA interference (RNAi) pathway. In fact, mutations in genes encoding for factors involved in RNAi (such as Dicer, Argonaute and RNA-dependent RNA polymerase (RdRP)) result in defects in heterochromatin assembly. Furthermore, H3K9 methylation has been implicated in regulating heterochromatic silencing during RNAi-mediated TGS. 49 Most strikingly, siRNA-mediated TGS in the same organism has also been shown to require RNA polymerase II, suggesting that transcription of the homologous target is required to initiate TGS. 50 This system is unlikely to be universally applicable, because many organisms, including Drosophila and mammals, seem to lack an RNA-dependent RNA polymerase. 51 Nevertheless, some RdRP-independent mechanisms may be active in mammals, as siRNA-mediated TGS has been shown also in human cells (reviewed in Ref. 52). While in some settings the ncRNA itself is directly involved in gene silencing or activation, in other instances, non-coding transcription, rather than the resulting transcript, is thought to serve a regulatory function. Non-coding transcription generally correlates with locus activation and is thought to be involved in the establishment of open chromatin domains, even though it has also the potential to mediate gene repression through a process of transcriptional interference. 6 Abarrategui and Krangel 53 analysed the significance of transcription through the murine Tcrα locus by introducing a transcription terminator downstream of the promoter. This approach allowed them to discriminate the effects of promoter activation from those depending on transcription per se, and they could show that transcription in this particular locus controlled chromatin structure and promoted the activation of immediately downstream Jα promoters, while repressing the activity of more distal promoters. Even though the basis for this differential effect is unclear, this work showed that non-coding transcription can have both positive and negative influences on downstream promoter activity and chromatin structure. A number of publications have now established a link between the ncRNA family of miRNAs and the development and regulation of cells of the immune system. MiRNAs are short ncRNAs involved in post-transcriptional gene silencing (PTGS) processes. Mature miRNAs are short (~22 nt), single-stranded RNAs that repress mRNAs post-transcriptionally by binding to partially complementary sites in the 3′ untranslated region of their target mRNA. Mature miRNAs are cleaved from ~70 nt hairpin structures called precursor miRNAs (pre-miRNAs) by the enzyme Dicer. Pre-miRNAs are in turn excised from a primary miRNA (pri-miRNA) transcript by the RNase III Drosha. Pri-miRNAs are typically transcribed by RNA polymerase II, 54 and seem to possess promoter and enhancer elements that are similar to those of protein-coding genes. 55 They can be thousands of nucleotides long and contain multiple pre-miRNAs, and can be spliced or unspliced. However, pre-miRNAs can also be contained within introns of protein-coding genes. The predominant regulatory effect of miRNAs is to repress their target mRNAs; mechanisms of repression include translational repression, mRNA cleavage, mRNA deadenylation and alteration of mRNA stability (see Refs 56 and 57 for some recent reviews on the topic). Differentiation in the immune system is controlled or modulated by an intricate network of growth and transcription factors that simultaneously regulate the commitment, proliferation, apoptosis, and maturation of progenitor cells. MiRNAs therefore appear ideally suited to rapidly adjust protein concentrations in cells, as would be expected to be required during cell differentiation, and accordingly, certain miRNAs are expressed in a stage-specific fashion in the hematopoietic system. 58–60 Two groups recently reported the generation of miR-155 deficient mice, one of the first examples of a microRNA deletion reported in the mouse. 61,62 In these mice, the function of miR-155 deficient T and B lymphocytes, as well as dendritic cells was impaired, resulting in immunodeficiency. These studies also demonstrated that miR-155 is essential for controlling specific differentiation processes in the immune response. Another striking example of the important role of specific miRNAs in the development and function of the immune system was provided by Li et al. 63 They demonstrated that TCR sensitivity and signalling strength can be modulated at the post-transcriptional level by miR-181a. In fact, miR-181a expression was higher in immature T-cell populations that recognize low-affinity self-antigens, such as double positive (DP) thymocytes, but lower in the more differentiated T-cell populations such as Th1 and Th2 effector cells that are only reactive to relatively high-affinity foreign antigens. Increasing miR-181a expression in mature T cells augmented their sensitivity to antigens, whereas inhibiting miR-181a expression in immature T cells reduced their sensitivity and impaired T-cell selection. Recently, Xiao et al. showed that miR-150 controls the expression of the transcription factor c-Myb in B lymphocytes in vivo, in a dose-dependent manner. 64 Alterations of miR-150 and, consequently, of c-Myb levels dramatically affected B lymphocyte development and responses. In particular, in striking contrast to mice with a B-cell specific deletion of c-Myb (in which B1 cells are absent), splenic B1 cells were expanded fourfold in number, while the development of T cells, follicular B cells, and marginal zone B cells was normal. These results support the concept that the physiological function of a given miRNA may lie in the control of the concentrations of just a few critical target proteins in a particular cellular context. While bioinformatics analyses predict hundreds of target genes for each miRNA, 65 only a few of these may represent targets with a significant biological function for a particular biological process. It is also noteworthy that miR-150 ectopic expression in non-hematopoietic cell lineages didn't show any obvious adverse physiological effect. Thus, miRNA-mediated control can also display specificity in terms of functional restriction to a particular differentiation pathway. It is worth of note that several viruses express both long (more than 100 nt) ncRNAs as well as miRNAs, with functions still largely unclear. The interaction of viruses with the mammalian RNA interference pathway has been discussed in some excellent reviews; 66–68 here, we will highlight a few recent advances in the field. The human cytomegalovirus (HCMV) expresses ncRNAs that allow infected cells to evade innate immune responses. Within hours of HCMV infection, a ~2·7 kb viral ncRNA (β2·7) accumulates and binds to components of the mitochondrial respiratory chain complex I, stabilizing the complex function. 69 This ultimately prevents premature apoptosis of infected cells, and also ensures the stable production of ATP during the viral life cycle. In another study, it was found that one HCMV miRNA, miR-UL112, inhibits the translation of the cellular protein MHC class I-related chain B (MICB), a ligand whose receptor is found on natural killer (NK) cells. 70 The resulting absence of MICB protein on the surface of HCMV-infected cells protects them against lysis by NK cells. This is a striking example of a novel miRNA-based immunoevasion mechanism exploited by HCMV, in which a viral miRNA directly down-regulates a host immune defense gene. Recently, it has also been shown that a viral miRNA can act as an orthologue of a cellular miRNA. 71 Specifically, miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155 and regulates an analogous set of mRNAs. At this point, it is unclear which of the many genes regulated by miR-155 and miR-K12-11 provide a replicative advantage to KSHV, but given the apparent role of miR-155 in the development of B-cell tumours, 72,73 miR-K12-11 expression in latently KSHV-infected B cells may contribute to the increased incidence of B-cell tumours seen in KSHV-infected patients. While the existence of viral ncRNAs is well established for DNA viruses like Herpesviruses, 66 the issue is still controversial for RNA viruses, like human immunodeficiency virus (HIV). 67,74–76 Nevertheless, it has been shown that some cellular miRNAs contribute to HIV-1 latency in resting T cells. 77 The latency of HIV-1 in resting primary CD4 T cells is the major barrier for the eradication of the virus in patients on suppressive highly active antiretroviral therapy. It now appears that host cell miRNAs expressed in resting T cells potently inhibit HIV-1 production, suggesting that manipulation of cellular miRNAs could represent a novel approach for purging the HIV-1 reservoir. Overall, it is becoming clear that not only many viruses encode they own regulatory ncRNAs to counteract immune responses, but host-encoded miRNAs have been also shown to interfere both positively and negatively with the viral life cycle. 68 It will be interesting to see what roles the many ncRNAs with still unknown functions play in virus-host interactions. Gene expression is regulated at many levels, all of which must ultimately be studied together to obtain a complete picture of gene regulatory networks. Genome-wide studies indicate that most of the non-protein coding sequences in the genome are actually transcribed and that even regions which lack sequence conservation can have epigenetic functions. The process of non-coding RNA transcription clearly participates in regulating the expression of protein-coding mRNAs, and the ncRNAs themselves contribute to the complex network needed to regulate cell function. We have only begun to understand how the cells use these regulatory ncRNAs for various aspects of gene expression ( 6; http://www.ensembl.org). Undeniably these findings suggest that epigenetic mechanisms and RNA plays a much broader and more profound role in the cell than previously envisioned. Many open questions and directions for future research remain: it's possible that more layers of genetic control are yet to be discovered, and their identification and understanding will be essential to elucidate normal as well as pathogenetic biological processes. We apologize to our colleagues whose outstanding contributions to this rapidly growing field were not cited as primary references because of space constrains. We would like to thank Anjana Rao for helpful discussions, support and critical reading of the manuscript, as well as Martha Neagu and Thomas Pertel for discussions and suggestions. MEP is a postdoctoral fellow in the laboratory of Anjana Rao and is supported by a fellowship from the NCI F32 CA126247-01. 1. Groth A, Rocha W, Verreault A, Almouzni G. Chromatin challenges during DNA replication and repair. Cell. 2007;128:721–33. [PubMed] 2. Krangel MS. T cell development: better living through chromatin. Nat Immunol. 2007;8:687–94. [PubMed] 3. Fraser P, Bickmore W. Nuclear organization of the genome and the potential for gene regulation. Nature. 2007;447:413–7. [PubMed] 4. Meaburn KJ, Misteli T. Cell biology: chromosome territories. Nature. 2007;445:379–781. [PubMed] 5. Birney E, Stamatoyannopoulos JA, Dutta A, et al. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature. 2007;447:799–816. [PubMed] 6. Prasanth KV, Spector DL. 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wide and locations of chromatin accessibility
