In their paper, “Hot L1s account for the bulk of retrotransposition in the human population,” Brouha et al. allege that only six highly active L1 elements account for most of the L1 retrotransposition activity in humans. LINE-1 elements (L1s) are autonomous non-LTR retrotransposons (they carry their own reverse transcriptase and are not flanked by long terminal repeats (LTRs)). At least 17% of the human genome is comprised of L1 elements, although the majority of these elements are severely 5’ truncated due to either a nonprocessive reverse transcriptase or to a unique crossover mechanism described elsewhere. Many other elements are internally deleted, rearranged, or otherwise nonfunctional. The full-length L1 element is 6 kb and has two open reading frames (the first is an RNA-binding protein with nucleic acid chaperone activity, and the second is an endonuclease and reverse transcriptase) flanked by UTRs. When the ORFs are transcribed into ORF1p and ORF2p, these proteins display cis preference; that is, they act on the mRNA that endoded them. Any disruption in the open reading frames is considered inactivating, although there is little evidence for this. L1 elements are traditionally thought to only retrotranspose in germ cells, although recent studies have challenged this. The L1 element is highly active, with one new L1 element present in the genome of one of every 10-40 people. In fact, each normally fertile male produces about
500 - 5000 sperm per minute with new L1 insertions. Because of this mutation rate, it is possible to follow the genetic drift of the L1 element throughout history as it gains and loses mutations, especially in the 3’ end. Many L1 elements have been newly identified when they have disrupted an important gene, often on the X chromosome in a male, and have caused a disease. Also of note, many other organisms have LINE-1 or similar elements and these elements have measurably contributed to speciation.
In this paper, Brouha et al. describe a dual bioinformatics and experimental analysis of
L1 elements in the human genome. They first discover that only 90 L1 elements in the genome are both full-length and have intact open reading frames. They demonstrated that nearly half of these L1s are polymorphic in humans, and cloned nearly all of the L1s to find that only half are active in a standard retrotransposition assay. They predict that there are 80-100 retrotransposition-competent L1 elements in the average human genome, but that six of those elements are highly active and contribute over 80% of all

retrotransposition activity. They conclude that these six “hot” L1s contribute nearly all of the in vivo retrotransposition activity, with the rest playing a smaller role in genome plasticity. They perform phylogenetic analysis on their L1 elements and concluded that the youngest L1 elements were both more likely to be active in their cultured cell assay but were also most polymorphic in the general population, as expected for active elements. Interestingly, they conclude that between one in two and one in 33 people harbor a new polymorphic L1 element. In summary, they conclude that they have doubled previous estimates for the number of active L1 elements in the human genome and that a small number of hot L1s accounts for the bulk of transposition.
Strengths :
1.
The authors compare their highly active L1 elements to the five known L1 elements that cause disease, in a cell culture assay, and find that their predicted active elements are in fact as active or more active as the diseasecausing elements, at least in cell culture.
2.
Figure 1 is one of the most intuitive, brilliant, and beautiful figures that I have ever seen in a scientific presentation. It conveys a massive amount of information in a small space and is displays its message as clearly as the best figures that I have seen.
3.
The authors nicely describe their methods, although the initial database search methods should probably not be relegated to supplementary methods. The authors also nicely describe the phylogenetic tree construction for their dataset.
4.
The authors point out that they used a limited number of genomes to come to their conclusion and that assaying more genomes would give a better idea of the extent of polymorphism of these elements.
5.
The authors perform sequence analysis to show that the further a sequence is from the “hot” consensus, the less likely it is to be active. This makes intuitive sense and it confirms their hypothesis that certain L1 sequences work better than others in retrotransposition.

6.
The authors use two independent methods to estimate the number of functional L1 elements in the genome and they are in good agreement.
Weaknesses:
1.
The authors mention only in passing that noncanonical L1 elements can be highly active, and this is also relegated to the supplemental methods when it probably deserves at least a sentence or two, if not a more detailed exposition.
2.
While the authors address in the discussion (and rather cursorily) the issue of the elements’ location in the genome, it could be incredibly useful to see more detail on this subject; for example, if the elements are all clustered or are distributed in similar compartments, it could be biologically interesting and very informative.
3.
The authors argue that there may be more active L1s in the entire population than there are inactive L1s, even though in any given person the inactive L1s probably dominate. Their argument is fairly weak and should not be included in the final paper.
In summary, this is an excellent paper and I recommend it for publication, with minor revisions. The research fits well with the goals of the journal and is easily worthy to be a
PNAS publication.