"Genetics:  LINEs in mind".

Eric M. Ostertag ^{1, 2} and Haig H. Kazazian, Jr ¹

¹ Department of Genetics and ² Department of Pathology, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104-6055, USA.
Email: kazazian@mail.med.upenn.edu

At least half the human genome consists of mobile elements, such as LINEs, some of which can jump around the genome. These elements have been crucial in genome evolution, but they may also contribute to human diversity.

Barbara McClintock won the Nobel Prize in Physiology or Medicine in 1983 for predicting the existence of mobile elements, pieces of DNA that move from one place in the genome to another. McClintock called them 'controlling elements' and proposed that they could account for developmental differences among individuals of a species — explaining, for example, the differences in maize-kernel colour that she observed [1]. Although her ideas were not well received at the time, they have proven to be remarkably prescient. On page 903 of this issue, Muotri et al. [2] provide evidence that mammalian mobile elements may have a role in creating "the uniqueness of individuals within a population".

Mobile elements, also called jumping genes, exist in all living things, but the 'long interspersed nucleotide element-1' (LINE-1, or L1 for short) is the only active human mobile element. This element encodes the machinery to move itself and to mobilize other elements [3]. Muotri et al. demonstrate that a human L1 is active in both cultured rat neural progenitor cells (NPCs) and the NPCs of a transgenic mouse. Remarkably, de novo L1 insertions in cultured NPCs sometimes alter the expression of neuronal genes, thereby affecting NPC differentiation (the process by which the cells mature into specialized cells).

If L1 insertion occurs in the NPCs of developing human neuronal cells, then some cells will contain de novo insertions. The authors cautiously speculate that activity of mobile elements might be responsible for creating some of the diversity among people. For example, if enough mobile DNA insertions occur in the brains of developing humans, then the outcome might be a change in their neuronal circuitry, for better or for worse.

One surprising find from this work is that L1 elements are actually active in NPCs. These elements are considered by many researchers to be no more than genetic parasites. From an evolutionary standpoint, L1 elements should be most successful if their expression is restricted to germ cells (sperm and oocytes), or to stem cells early in development, because mobility in these cell types will lead to an expansion in the number of L1 elements. Conversely, de novo L1 insertions in other cell types (somatic cells) cannot be passed on to future generations, and, if detrimental to host reproduction, would harm the chances of L1 propagation.

In fact, previous studies of L1 expression and mobility demonstrate L1 activity in germ cells and in early developmental cells [4, 5, 6], but not in other cell types. There has been only one previous example of L1 mobility in a human somatic cell: this insertion disrupts a gene that contributes to colon cancer [7]. Muotri et al. provide the first evidence of L1 activity in normal cells cultured directly from an animal sample, and the first evidence of somatic L1 activity late in development of a transgenic mouse.

The expression of L1s in NPCs appears to be inversely correlated with the expression of SOX2, a gene that is poorly expressed in developing NPCs but that has several vital functions in adult neural cells. The authors further demonstrate that L1 activation is related to changes in histone proteins that are associated with gene expression in general. Histones interact directly with DNA, and their acetylation and methylation pattern can determine whether a region of DNA is 'open' and transcribed [8]. That histone modification might be a host mechanism to control L1 activity is an intriguing possibility.

Also a surprise is the extent to which de novo L1 insertions affect NPC development. An analysis of mammalian genomes demonstrates that L1 elements insert more or less randomly, landing both within and outside genes [9, 10]. The authors have not analysed enough de novo insertions from cultured NPCs to achieve statistical significance, but there seems to be a striking preference for insertions into or near genes. This particularly applies to genes that are neuronally expressed — in two cases (out of 17) insertions occur in the same neuronally expressed gene. Even more interesting is the finding that NPC differentiation is affected by L1 insertions. The researchers convincingly demonstrate that one such insertion into the Psd-93 gene increases the expression of that gene, which in turn induces neural differentiation.

Why is there a discrepancy between the random insertion pattern of L1 elements previously found in the genome, and the apparent non-random insertion pattern observed in cultured NPCs? One possibility is that 'open' areas of DNA are more accessible for L1 insertion, and perhaps there are fewer 'open' areas in NPCs than in germ cells. Neuronally expressed genes are by definition 'open' in neuronal cells, and may be preferred sites for new insertions. Future experiments should determine whether the non-random insertion pattern also occurs in vivo or is peculiar to cultured cells.

Muotri et al. [2] hypothesize that mobile elements could affect neuronal development and diversity of brain function in humans, but there are several questions to be answered before this can be accepted. First, is the frequency of de novo insertions high enough to affect neuronal function? Most NPCs are not expected to contain de novo insertions, and many NPC insertions will have no effect on neuronal function (Box 1). 

It is impossible to determine directly the frequency of insertions that occur in vivo in human neuronal precursor cells (NPCs), and Muotri et al. [2] were not able to quantify this number in transgenic mice. It seems, from stained brain sections, that insertions do not occur more often than once in 10 cells, perhaps only occurring as rarely as once in 1,000 cells. Although a small fraction of cells may appear to have new insertions, it is difficult to extrapolate the activity of an unknown number of highly active L1 elements in a transgenic mouse to the activity of a number of endogenous L1 elements. Most insertions will not occur in genes. If L1 insertion is entirely random, the percentage of insertions into genes would be about 30% — the percentage of the genome that is made up of genes (introns plus exons). Muotri et al. [2] have reason to believe that the percentage of gene insertions may be higher than 30% because of non-random insertion. However, to affect neural function, an insertion must occur in a neuronally expressed gene, and the insertion must have an effect on cell fate even when only one of the two copies of a gene is disrupted. Insertions into a single copy of some neuronally expressed genes may have no effect.

Considering the predicted rarity of these events in vivo, the effects of mobile elements on neurons may be limited. That said, the authors did find several examples of these events in cultured cells.

Second, is the ability of L1 to move in NPCs a random quirk of nature? Or is this an evolutionarily maintained mechanism that creates individual variation? At first glance, it would seem that this process cannot be evolutionarily maintained. Any insertions that occur in NPCs are not passed on in the germ line, so any changes that insertions make to an individual are eliminated with each generation. However, if the mechanism to create diversity is encoded in the germ line (for example, by the number and activity level of mobile elements), and if diversity is favoured by natural selection, then this mechanism can be maintained through evolution.

Time and further research will determine whether McClintock's hypothesis that mobile elements have a significant role in an organism's development can be extended from maize to humans, and specifically to the function of human neurons.

References:

1. McClintock, B. Yb. Carnegie Inst. Wash. 48, 142-154 (1949).

2. Muotri, A. R. et al. Nature 435, 903-910 (2005).

3. Dewannieux, M., Esnault, C. & Heidmann, T. Nature Genet. 35, 41-48 (2003).

4. Branciforte, D. & Martin, S. L. Mol. Cell Biol. 14, 2584-2592 (1994).

5. Ostertag, E. M. et al. Nature Genet. 32, 655-660 (2003).

6. Prak, E. T., Dodson, A. W., Farkash, E. A. & Kazazian, H. H. Jr Proc. Natl Acad. Sci. USA 100, 1832-1837 (2003).

7. Miki, Y. et al. Cancer Res. 52, 643-645 (1992).

8. Turner, B. M. Cell 111, 285-291 (2002).

9. Lander, E. S. et al. Nature 409, 860-921 (2001).

10. Waterston, R. H. et al. Nature 420, 520-562 (2002). 

1. Song X, Sun Y, and Garen A, "Roles of PSF protein and VL30 RNA in reversible gene regulation".

2. Muotri AR, Chu VT, Marchetto MCN, Deng W, Moran JV, and Gage FH, "Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition".

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