The mouse genome was sequenced in 2002 as a primary model in which to study gene function and human diseases and to develop drugs1. This was followed by maps of transcribed messenger RNA molecules and of long, non-protein-coding RNAs, which facilitated such experiments and analysis2. Yet although 17 mouse strains have been sequenced3, genome function and regulation cannot be understood by sequence analysis alone. Now, in four papers published in this issue4,5,6,7, the Mouse ENCODE Consortium presents data sets that dramatically enhance our understanding of the regulation of the mouse genome, and of the similarities and differences compared with the human genome.
The ENCODE project8,9 was started by the National Human Genome Research Institute in 2003, with the aim of mapping functional elements of the human genome. The project, later expanded as Mouse ENCODE and modENCODE (to include invertebrate model organisms), has driven technology development and standardization for the identification of expressed RNAs and regulatory regions. These technologies have given rise to comprehensive data sets for analysing genome regulation and comparing this across species. Among the resources are libraries of mRNA sequences and maps of genomic regions that are bound by transcription factors or by RNA polymerases (the enzymes that initiate RNA transcription). There are also data sets on chemical modifications to the histone proteins around which DNA is wrapped (forming a complex called chromatin). Such modifications alter the accessibility of the DNA to other proteins and thereby demarcate transcriptionally 'active' or 'repressed' chromatin regions. And there are data on large-scale chromatin and chromosome structures.
The Mouse ENCODE Project has taken advantage of the ENCODE experience to provide a much-needed comprehensive resource for mouse genomics and its first in-depth analysis. Stergachis and colleagues' data5 (page 365) reveal that, in the roughly 75 million years of evolution since humans and mice diverged, the primary (nucleic-acid) sequence of regulatory elements has changed dramatically. About half of the transcription-factor binding sites in regulatory elements of the mouse genome are not present in the equivalent (orthologous) elements in humans, and around one-quarter of them have migrated to different positions (Fig. 1). Regulatory elements that are distant from the gene that they regulate (enhancers) have diverged more than those that are close (promoters). Despite this divergence, Cheng et al.6 (page 371) show that there is similar chromatin activity in orthologous promoter regions in the two genomes, suggesting that different transcription factors could be used to achieve similar transcriptional activity. Furthermore, despite the different primary sequences of many regulatory elements, the basic reciprocal regulatory networks among transcription factors are evolutionarily conserved between mice and humans5.
Surprisingly, the Mouse ENCODE Consortium (Yue et al.4; page 355) finds that sequences commonly considered useless or harmful, such as retrotransposon elements (stretches of DNA that have been incorporated into chromosomal sequences following reverse transcription from RNA), have species-specific regulatory activity. Because retrotransposon elements can contain embedded transcription-factor binding sites, this may provide unexpected regulatory plasticity (Fig. 1). Evolutionary conservation of primary sequence is typically considered synonymous with conserved function, but this finding suggests that this concept should be reinterpreted, because insertions of retrotransposon elements in new genomic regions are not conserved between species.
Although gene expression might be expected to be similar in the same organs and tissues in different species, comparative analyses by the consortium4 reveal that the expression level of many genes (but not all gene categories) is species specific, rather than organ specific. These differences may derive from the fact that organs are composed of different cell types in mouse and human tissues, but it is more likely to have arisen from different basic transcriptional activity driven by different regulatory elements.
Despite these variations between the mouse and human genomes, Cheng et al.6 show that many single-nucleotide sequence differences that have been associated with diseases in genome-wide association studies in humans are localized to orthologous regions of the mouse genome that have modifications that mark active chromatin. This finding validates the importance of the mouse as a model organism for ongoing disease studies.
Finally, Pope et al.7 (page 402) have generated high-quality maps of the physical position of chromosomes in the nuclei of mouse and human cells. These maps show that the boundaries of replication domains (genomic regions that replicate at the same time during cell division) correlate well with topologically associating domains — chromosome structures that are associated with the regulation of gene expression.
Analysis of these data will continue, both broadly and in the context of specific biological questions, although new tools for visualizing, analysing and interpreting such data are needed to open them up for broader use by experimental biologists. But the existing findings are already thought-provoking. For example, they suggest that we should rethink the relationship between genomic function and evolutionary conservation. Regulatory regions and long non-coding RNAs (lncRNAs) are not subject to the evolutionary constraints of protein-coding genes, which may help to explain the sequence drifts reported in these papers. However, it is striking that transcription-factor networks are conserved despite low conservation of their binding positions in the genome. Further experiments are needed to establish whether transcription-factor interactions with regulated regions always promote transcription or whether they can also be repressive. The differences in regulation between mice and human genomes that have emerged from these studies should all be taken into account when using mouse models to assess biological functions and, in particular, drug responses.
Some genomic features in particular, such as lncRNAs, warrant further investigation. The Mouse ENCODE Project analysed only RNA molecules that are polyadenylated (they have a string of adenine bases at the 3′ end); although this modification marks most mRNAs, many lncRNAs are not polyadenylated10, and so analysis of non-polyadenylated RNAs in mice will be needed to better define the similarities and differences between the full complement of RNA transcripts in mice and humans. A comprehensive map of orthologous human and mouse lncRNAs will also be useful for experimental tests of the function of human lncRNAs in mice.
Furthermore, there is room to expand the data set on transcription-factor binding sites generated by Cheng and colleagues6, because their experiments were performed using mouse cells that are easy to cultivate (MEL and CH12) and thus provide plenty of experimental material, but they do not represent the biological variability present in the hundreds of cell types found in mammals11. It will also be useful to replicate these studies in different mouse strains and to connect differences in genome sequence3 between the strains to differences in gene regulation and traits.
The data sets provided by the mouse ENCODE project boost our capacity to analyse the mouse genome in a way that was unthinkable a decade ago, and allows us to gain insights into dimensions that were not foreseeable. Understanding genomic regulation in mice is much more than a linear addition to our knowledge of genome regulation overall — it is an essential step towards better understanding human biology and improving biomedical applications and drug development.
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