MicroRNAs regulate "de novo" DNA methylation and histone mRNA 3' end formation in mammalian cells

MicroRNAs (miRNAs) are known to have many important functions in mammalian cells.
They can influence the expression of their target genes and in this way regulate the
function of not only their primary targets, but also of the pathways and mechanisms
acting downstream of the primary targets. There are several key proteins that are required
for the biogenesis of miRNAs and for mediating the repressive functions of miRNAs in
mammals, the most critical being the ribonuclease (RNase) III enzyme Dicer. Since Dicer
is required for generation of all known mammalian miRNAs, depletion of Dicer is an
appealing strategy to identify and study the pathways under miRNA-mediated control.
Deletion of Dicer in mouse embryonic stem cells (ESCs) is rendering the cells to
slow growth rate and inability to differentiate, and thus, to loose their most important
feature i.e. pluripotency. We aimed to understand in further detail the causes behind these
critical defects. We have performed transcriptional profiling of Dicer-deficient ESCs and
through bioinformatic analysis we identified miRNAs of the ESC-specific miR-290
cluster to be functionally most important for mouse ESCs. These miRNAs were found to
directly control the expression of several hundred primary targets and through their
regulation influence many features of the ESCs. We found the miR-290 miRNAs to
contribute to the growth rate of the ESCs and to influence also expression of many
secondary target genes. Among their secondary targets we identified de novo DNA
methyltrasferases (DNMT3s) that were significantly downregulated in Dicer-deficient
mouse ESCs. The downregulation was due to an increased expression of Retinoblastomalike2
(RBL2), a transcriptional repressor and primary target miR-290 miRNAs. As a
consequence of lowered DNMT3 expression the cells were unable to methylate DNA at
the promoter of pluripotency genes such as Oct-4 (Octamer-binding transcription factor-4,
also known as Pou5f1 for POU-domain, class 5, transcription factor 1), and thus,
incapable of fully silencing these genes during differentiation. Hence, regulation of
DNMT3s by miR-290 miRNAs is contributing to the maintenance of mouse ESC
pluripotency.
Further analysis of the promoter of primary miR-290 transcript (pri-miR-290)
showed that the ESC specific expression and subsequent silencing of the transcript during neuronal differentiation is regulated by the chromatin status of the promoter. During
neuronal differentiation the pri-miR-290 promoter looses histone modifications
characteristic of active genes and gains typical marks of silenced chromatin. This is
followed by de novo DNA methylation of the pri-miR-290 promoter. It is likely that the
silencing of pri-miR-290 depends on DNA methylation of its promoter, thus allowing an
auto-regulatory loop between the miRNAs and DNMT3 enzymes.
In addition to Dicer-deficient mouse ESCs, we have studied the importance of
Dicer as well as Argonaute proteins for the function of human cell lines by inducibly
depleting these proteins in human HEK293T-REx cells. We observed that an intact RNA
silencing pathway is needed for normal expression of many of the replication-dependent
histone genes. We found up to 25% of all histone mRNAs to be upregulated upon loss of
RNAi machinery and more detailed analysis of one of the histone genes, HIST1H3H,
demonstrated that the upregulation was due to enhanced polyadenylation of the histone
mRNA. This is in contrast to the normal 3’ end processing of replication-dependent
histone mRNAs that takes place at the 3’ end-proximal stem-loop and is not followed by
polyadenylation. The analysis of RNA from Dicer- or Dgcr8-deficient ESCs showed that
this type of regulation of 3’ end formation by RNA silencing pathway is conserved in
mice and depends on the generation of miRNAs. Thus, miRNAs seem to regulate the 3’
end processing of replication-dependent histone mRNAs. Future work will be needed to
identify specific miRNAs and processing factors involved.