Referee 1's review:

This manuscript addresses the role LSD1 in the transcriptional repression of hTERT, which is strictly repressed in most somatic cells, but activated in the majority of cancer cells. The issue of hTERT regulation is an important one, because telomerase activity, which is predominantly regulated by hTERT expression, is thought to be a critical determinant of cancer proliferation. The factors that contribute to hTERT repression and/or activation are only partly understood.

Because H3-K4 methylation of the hTERT promoter induces hTERT expression, the authors hypothesized that H3-K4 demethylation may be required for hTERT repression and that LSD1, a histone demethylase with specific activity for methylated H3-K4, may be responsible for maintaining H3-K4 demethylation at the hTERT promoter.

Overall, the data presented in this well-written manuscript support their hypothesis. They demonstrate local effects of LSD1 on hTERT H3-K4 methylation (as opposed to global effects) and demonstrate LSD1 recruitment to the hTERT promoter. While LSD1 inhibition or down regulation had an effect on hTERT expression, it was a minor one, although this is clearly stated in the manuscript. A more robust synergistic effect was observed with HDAC inhibition. The fact that LSD1 inhibition or knockdown still led to increases in hTERT mRNA expression in the cancer cell lines would suggest that it is not one of the required switches for hTERT activation in telomerase positive malignant cells, minimizing the significance of this work to cancer biology.

Nonetheless, the observations and conclusions should be included in the scientific literature as they are likely to be of interest particularly to those in specialized areas such as chromatin remodeling and telomerase regulation. Specific criticisms are noted:
1) The abnormally migrating band in Figure 1B lane 3 (-LSD1, +CoREST SiRNA) needs to be explained.

2) Minus standard deviation bars need to be included in Figure 1D and whether there is a statistical difference in relative luciferase activity at the various levels of tranylcypromine should be determined.

3) It is not clear from the gels that there was a dose-dependent significant upregulation of hTERT expression in Figure 2A. In some cases, there appears to be no effect at 10 μM (SiHa and SW756) and in others less expression at 100 μM relative to 10 μM (HeLa).

Quantitative real-time PCR would allow them to determine whether there are indeed dosedependent significant effects. Otherwise the terms dose-dependent and significant should be removed.

4) Similarly, it does not appear that the 100 μM tranylcypromine treatment had an effect on HeLa cell telomerase activity. This should be stated in the text and may relate to the possibility that hTERT mRNA was actually not upregulated at this dose level (see above).

Minor points:
1) Figure 1C should be noted in the initial description of the LSD1 siRNA knockdown (in paragraph 1 of the Results section).

2) The siRNA data for the A549 and SW756 cells should be mentioned in the results section if the data are to be included in the manuscript.

3) The last sentence of the second paragraph on page 8 should be restated to note the effects were observed in cervical cancer cell lines. Similarly, the final sentence of the first paragraph of the discussion should read … normal primary and cancer human cell lines.

4) The control and DMSO lanes should be specified on Figure 4A.

5) The ChIP data are quite convincing. A description of the location of the primers within the Results section as opposed to the Materials and Methods would be helpful.

6) Materials and Methods are presented in sufficient detail.

----------
N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.