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The findings that an RNA is not necessarily either coding or non-coding, or that a precursor RNA can produce different types of mature RNAs, whether coding or non-coding, long or short, have challenged the dichotomous view of the RNA world almost 15 years ago. Since then, and despite an increasing number of studies, the diversity of information that can be conveyed by RNAs is rarely searched for, and when it is known, it remains largely overlooked in further functional studies. Here, we provide an update with prominent examples of multiple functions that are carried by the same RNA or are produced by the same precursor RNA, to emphasize their biological relevance in most living organisms. An important consequence is that the overall function of their locus of origin results from the balance between various RNA species with distinct functions and fates. The consideration of the molecular basis of this multiplicity of information is obviously crucial for downstream functional studies when the targeted functional molecule is often not the one that is believed.

DNA methylation mostly occurs within the context of CpG dinucleotides and is essential for embryonic development and gene repression. It is generally accepted that DNA methyltransferases carry out specific and non-overlapping functions, Dnmt3a and Dnmt3b being responsible for the establishment of methylation around the time of implantation and Dnmt1 ensuring that methylation is faithfully copied to daughter cells via what has come to be known as "maintenance methylation." This longstanding view has been challenged over the years with the observation that Dnmt1 alone is incapable of perfect maintenance methylation. A new model is emerging that takes into account a contribution of the de novo enzymes Dnmt3a and Dnmt3b in the maintenance of the DNA methylation. We recently showed that certain germ line genes are specific targets of Dnmt3b, and that Dnmt3b remains bound to their promoter regions in somatic cells via interaction with the transcriptional repressor E2F6. It is tempting to consider an ongoing role for Dnmt3b in the methylation of germ line genes in somatic cells. We propose here observations in support of the hypothesis that the maintenance of methylation and subsequent silencing of a handful of germ line genes requires Dnmt3b but not Dnmt1. In addition to suggesting a new role for Dnmt3b in the protection of somatic cells against the promiscuous expression of the germ line program, these observations are of particular interest in the field of carcinogenesis, given that the expression of catalytically inactive Dnmt3b isoforms and aberrant expression of germ line genes are commonly observed in cancer cells.

The heterochromatin-associated H3K9 tri-methylase Suv39h1 is involved in the permanent silencing of E2F target genes in differentiating but not in quiescent cells. Here, we tested the hypothesis that permanent silencing of E2F target genes is associated with their subnuclear positioning close to the pericentromeric heterochromatin compartment, enriched in Suv39h1. Using fluorescence in situ hybridization, we analyzed the subnuclear localization of three E2F target genes relative to the pericentromeric heterochromatin, in cycling fibroblasts or differentiating myoblasts. We observed that all three E2F-target genes have a tendency to relocate closer to the pericentromeric heterochromatin, only when cells differentiate and undergo an irreversible cell cycle withdrawal. These data suggest that repression of E2F target genes in cycling or in differentiating cells is achieved through distinct mechanisms. In differentiating cells, permanent silencing is driven by a Suv39h1-dependent H3K9 tri-methylation and positioning close to the heterochromatin compartment, whereas repression in cycling cells seems independent from subnuclear positioning and requires distinct H3K9 methylation levels.

A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.