Phillip Grote

Mechanisms of long noncoding RNA function in development and disease

Since decades it has been known that non-protein-coding RNAs have important cellular functions. Deep sequencing recently facilitated the discovery of thousands of novel transcripts, now classified as long noncoding RNAs (lncRNAs), in many vertebrate and invertebrate species. LncRNAs are involved in a wide range of cellular mechanisms, from almost all aspects of gene expression to protein translation and stability. Recent findings implicate lncRNAs as key players of cellular differentiation, cell lineage choice, organogenesis and tissue homeostasis. Moreover, lncRNAs are involved in pathological conditions such as cancer and cardiovascular disease, and therefore provide novel biomarkers and pharmaceutical targets. Here we discuss examples illustrating the versatility of lncRNAs in gene control, development and differentiation, as well as in human disease.

Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6

The vertebral column and skeletal muscles of vertebrates are derived from the paraxial mesoderm, which is laid down initially as two stripes of mesenchymal cells alongside the neural tube and subsequently segmented. Previous work has shown that the wingless‐type MMTV integration site family (WNT), fibroblast growth factor‐ and Delta–Notch signalling pathways control presomitic mesoderm (psm) formation and segmentation. Here, we show that the expression of mesogenin 1, a basic helix–loop–helix transcription factor, which is essential for psm maturation, is regulated by synergism between WNT signalling and the T‐box 6 transcription factor, involving a feed‐forward control mechanism. These findings emphasize the crucial role of WNT signalling in the control of psm formation, maturation and segmentation.

The long non-coding RNA Fendrr links epigenetic control mechanisms to gene regulatory networks in mammalian embryogenesis

Epigenetic control mechanisms determine active and silenced regions of the genome. It is known that the Polycomb Repressive Complex 2 (PRC2) and the Trithorax group/Mixed lineage leukemia (TrxG/Mll) complex are able to set repressive and active histone marks, respectively. Long non-coding RNAs (lncRNAs) can interact with either of these complexes and guide them to regulatory elements, thereby modifying the expression levels of target genes. The lncRNA Fendrr is transiently expressed in lateral mesoderm of mid-gestational mouse embryos and was shown to interact with both PRC2 and TrxG/Mll complexes in vivo. Gene targeting revealed that loss of Fendrr results in impaired differentiation of tissues derived from lateral mesoderm, the heart and the body wall, ultimately leading to embryonic death. Molecular data suggests that Fendrr acts via dsDNA/RNA triplex formation at target regulatory elements, and directly increases PRC2 occupancy at these sites. This, in turn, modifies the ratio of repressive to active marks, adjusting the expression levels of Fendrr target genes in lateral mesoderm. We propose that Fendrr also mediates long-term epigenetic marks to define expression levels of its target genes within the descendants of lateral mesoderm cells. Here we discuss approaches for lncRNA gene knockouts in the mouse, and suggest a model how Fendrr and possibly other lncRNAs act during embryogenesis.

DNA–Methylome Analysis of Mouse Intestinal Adenoma Identifies a Tumour-Specific Signature That Is Partly Conserved in Human Colon Cancer

Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.

Long noncoding RNAs in organogenesis: making the difference

A large proportion of the cellular transcriptome of higher vertebrates consists of non-protein coding transcripts, among them the long noncoding RNAs (lncRNAs). Although lncRNAs are functionally extremely divergent, many ncRNAs have been shown to interact with chromatin modifying complexes and/or with transcriptional regulators. Via such interactions, many lncRNAs are involved in controlling the activity and expression level of target genes, including important regulators of embryonic processes, and thereby fine-tune gene regulatory networks controlling cell fate, lineage balance, and organogenesis. Intriguingly, an increase in organ complexity during evolution parallels a rise in lncRNA abundance. The current data suggest that lncRNAs support the generation of cell diversity and organ complexity during embryogenesis, and thereby have promoted the evolution of more complex organisms.

An inducible RNA interference system for the functional dissection of mouse embryogenesis

Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.

Dlk1 Promotes a Fast Motor Neuron Biophysical Signature Required for Peak Force Execution

Quick, Quick, Slow The slow muscles of postural stability and the fast muscles of running and jumping are driven by motor neurons that are differentiated by fast and slow biophysical properties. By retrograde labeling of mouse and chick muscle fibers, Müller et al. (p. 1264) characterized the developmental distinctions between fast and slow motor neurons. A transmembrane protein, when over- or underexpressed, was discovered to drive specification of the motor neurons and a downstream effector specified some, but not all, of the biophysical attributes. The fast versus slow profile of motor neurons is controlled by expression of a membrane protein. Motor neurons, which relay neural commands to drive skeletal muscle movements, encompass types ranging from “slow” to “fast,” whose biophysical properties govern the timing, gradation, and amplitude of muscle force. Here we identify the noncanonical Notch ligand Delta-like homolog 1 (Dlk1) as a determinant of motor neuron functional diversification. Dlk1, expressed by ~30% of motor neurons, is necessary and sufficient to promote a fast biophysical signature in the mouse and chick. Dlk1 suppresses Notch signaling and activates expression of the K+ channel subunit Kcng4 to modulate delayed-rectifier currents. Dlk1 inactivation comprehensively shifts motor neurons toward slow biophysical and transcriptome signatures, while abolishing peak force outputs. Our findings provide insights into the development of motor neuron functional diversity and its contribution to the execution of movements.

Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1‐controlled polyamine export.

Roles for long non-coding RNAs in physiology and disease

While the vast majority of the genome is transcribed into RNA, only a small fraction of these transcripts have protein-coding potential. A large fraction of the transcribed RNA belongs to the class known as long non-coding RNAs (lncRNAs). Several recent studies have shown that at least some of these lncRNA transcripts represent functional RNA molecules. LncRNAs can utilize a wide range of mechanisms to regulate the RNA and/or the protein content of a cell on the transcriptional and the post-transcriptional levels. So far, many studies have identified differentially expressed lncRNAs in various physiological contexts, genetic disorders and human diseases. A steadily increasing number of studies could establish functional roles for some of these lncRNAs in developmental processes, cancer and tissue homeostasis. Taken together, these functions provide an additional layer of gene regulation and contribute to the high complexity of physiological and disease-related phenotypes.

The tissue-specific transcriptomic landscape of the mid-gestational mouse embryo

Differential gene expression is a prerequisite for the formation of multiple cell types from the fertilized egg during embryogenesis. Understanding the gene regulatory networks controlling cellular differentiation requires the identification of crucial differentially expressed control genes and, ideally, the determination of the complete transcriptomes of each individual cell type. Here, we have analyzed the transcriptomes of six major tissues dissected from mid-gestational (TS12) mouse embryos. Approximately one billion reads derived by RNA-seq analysis provided extended transcript lengths, novel first exons and alternative transcripts of known genes. We have identified 1375 genes showing tissue-specific expression, providing gene signatures for each of the six tissues. In addition, we have identified 1403 novel putative long noncoding RNA gene loci, 439 of which show differential expression. Our analysis provides the first complete transcriptome data for the mouse embryo. It offers a rich data source for the analysis of individual genes and gene regulatory networks controlling mid-gestational development.