Download We have found that an H3K27 methyltransferase activity co

1
Manuscript N°: ONC-2007-00554
The transcriptional repressor NIPP1 is an essential player inEZH2-mediated gene silencing
Response to Reviewers
We were pleased to learn that our manuscript could become acceptable for publication in
Oncogene provide we responded adequately to the comments, with additional data where
requested. We have addressed the criticisms point-by-point, as detailed below.
Reviewer 1
1. Although NIPP1 function is still unknown, it looks as a relatively pleiotropic regulator. It
would be important to clarify that Ezh2 regulation by NIPP1 is a direct event rather than an
indirect one. The fact that NIPP1 and eed, a Ezh2 partner in PRC2,3,4 complexes and needed
for their H3K27me3 activity, would support such a direct role. However, it is not possible to
ensure that all eed is Ezh2 associated, making desirable that an association between NIPP1
and Ezh2 is shown, ideally in non-transfected cells. Also, being Ezh2 activity regulated
postranslationally, for instance by Akt-dependent phosphorylation, the possible effects of
NIPP1 on Ezh2 modifications (since general stability seems not affected) could be looked
into. The same for NIPP1 association with nucleosomes.
Reply:
- We have found that an H3K27 methyltransferase activity co-immunoprecipitates with
endogenous NIPP1, and that endogenous NIPP1 and EZH2 co-immunoprecipitate, as detected
by immunoblotting. In addition, we found that NIPP1 is a direct interactor of both
recombinant EZH2 and EED, and we have mapped their distinct NIPP1 interaction sites. All
these data are part of a short manuscript that was submitted (the revised version will be
submitted on July 3rd) following the submission of the present manuscript to Oncogene. We
have now referred to these submitted data of ours (Roy et al., unpublished data) in the
Discussion section (p 10, para 2, line 8 and 10 and p11 line 1).
- new text at the end of p4 and top of p5
We have performed immunostainings with phospho-Ser21 specific EZH2 antibodies on
outgrowths of NIPP1wt and NIPP1-/- blastocysts (new Fig. 1d). The level of phosphorylation
of Ser 21 of EZH2 was unaffected in NIPP1-/- outgrowths (new Fig. 1d).
2. The claim that NIPP1 genomic targets overlap partially Ezh2 bound loci is not accurate.
There are a number of loci that bind Ezh2 (as seen from chromatin immunoprecipitation
experiments); however, they tend to be rather cell type specific. Therefore, comparing the set
of NIPP1 bound loci in Hela cells with sets of Ezh2 bound loci in other cell types is not ideal.
In this regard, I find bizarre the choosing of MYT1 nd MSMB, two Ezh2 targets (in prostate
cells), to test NIPP1 binding without knowing whether they were upregulated in the NIPP1
knockdown experiments (I didn't see them in Fig. 3b, but didn't check the supplementary
tables since they are very difficult to look up as genes are identified by their ID numbers,
which makes really cumbersome to make anything out of them. I'd imagine that a way out of
this would be to knockdown NIPP1 in a cell line in which a well established set of Ezh2
targets has been defined (ES cells are an example) and look at the effects on gene expression,
followed by ChIP experiments on these targets. A reasonable set of targets would suffice,
without having to do the genome-wide experiments again. Because toxicity in the NIPP1
knockdown (are all cells in Fig 2a center and right alive?) could be an issue, at least the
chromatin immunoprecipitation of known Ezh2 targets may be informative
Reply:
2
We accepted all these criticisms and have dealt with them as follows:
- adjusted Figure 5
We have removed the MSMB gene from Figure 5. MYT1 is as an established polycomb target,
since it was shown by many different groups and in different cell lines to be an EZH2 target.
Kirmizis et al., (2004) used the colon cancer cells SW480; Bracken et al. (2006) used the
human embryonic fibroblasts TIG3 and Viré et al. (2006) used U2OS, an osteosarcoma cell
line.
- new Fig. 6 and 7, new text on last paragraph on p7 , top of p8 and second paragraph at p9
We have generated a completely new Fig. 6 with a large set of likely Polycomb target genes.
We selected 21 genes that showed a significantly altered expression in PC-3 cells after
knockdown of NIPP1 and/or EZH2 and that were recently identified as likely PcG targets
genes because of trimethylation on H3K27 and association with Suz12 (Boyer et al., 2006;
Bracken et al., 2006; Lee et al., 2006). In addition we included in our analysis four
established EZH2 target genes and a negative control (Kirmizis et al., 2004; Bracken et al.,
2006; Viré et al., 2006, Beke et al., 2007). All these genes were grouped in eight classes,
based on their response in our NIPP1/EZH2 knockdown experiments. We performed ChIP
analysis with NIPP1, EZH2 and H3K27me3 antibodies on three genes in each class. Rabbit
anti-mouse immunoglobulines were used as a control. The data are consistent with our
proposal that NIPP1 is associated with a large set of Polycomb targets and these targets
overlap with EZH2 bound loci.
In the new Fig. 7, we selected five genes that were trimethylated on H3K27, each from a
distinct group, as defined in Fig. 6. The RNAi-mediated knockdown of either NIPP1 or EZH2
resulted in a loss of trimethylation of three out of these five genes, consistent with the notion
that both NIPP1 and EZH2 are required for the initiation and/or maintenance of H3K27
trimethylation.
- A new column was added in Tables S1 and S2 with the official gene names.
- adjusted legend of Fig. 2
The remaining cells following the RNAi of NIPP1/EZH2 were still alive, as shown by the
formation of purple formazan crystals in the MTT (tetrazolium salt) assay (not shown). This is
now indicated in the legend to the Fig. 2.
Minor remarks:
1. DAPI and Ezh2 staining of NIPP1 -/- blastocysts outgrowths (Fig 1a) are of bad quality
because of an inconvenient shadow.
Reply:
The stainings in Fig. 1a were repeated and the new panel is of an excellent quality, without
any shadow.
2. Fig 1D could be taken into supplementary material if at all since they are commercial
antibodies and it only shows that non histone proteins are not enriched in histone preps and
the other way around, that histone proteins are difficult to detect in histone poor preps.
Reply:
We have removed Fig. 1d, as requested.
3. I'd imagine that SIPP1 as a loading control is an accepted control isn't it.
Reply:
3
SIPP1 is a nuclear scaffold protein (mentioned in the legend of Fig. 3) and is structurally
unrelated to NIPP1. SIPP1 is a ubiquitously expressed protein and is therefore an accepted
loading control for nuclear proteins.
4. Fig 2b: y axis lacks a legend; SD bars correspond to how many experiments
Reply:
The data from Fig. 2b were obtained from three independent experiments (n=3). This is now
mentioned in the legend of Fig. 2. The legend to the Y-axis has been added.
5. Fig. 6, data correspond to what cell type?
Reply:
It is now mentioned in the legend that the experiments were done with PC-3 cells.
Reviewer 2
…Despite of these critiques, I still believe that discoveries in this study are novel and
worthwhile reporting in Oncogene if these points could be appropriately corrected. I therefore
suggest to investigate NIPP1- and Ezh2-binding and degree of H3K27 trimethylation at least
at three genes in six categories shown in Fig. 3c; genes up-or downregulated in exclusively
NIPP1 KD or EZH2 KD or in both, and unaffected. This may provide rather general view for
functional correlation between NIPP1 and PRC2.
Reply:
This point was well taken. We selected 21 genes that showed a significantly altered
expression in PC-3 cells after knockdown of NIPP1 and/or EZH2 and that were recently
identified as likely PcG targets genes because of trimethylation on H3K27 and association
with Suz12 (Boyer et al., 2006; Bracken et al., 2006; Lee et al., 2006). In addition we
included in our analysis four established EZH2 target genes and a negative control (Kirmizis
et al., 2004; Bracken et al., 2006; Viré et al., 2006, Beke et al., 2007). All these genes were
grouped in eight classes, based on their response in our NIPP1/EZH2 knockdown
experiments. As requested, we performed ChIP analysis with NIPP1, EZH2 and H3K27me3
antibodies on three genes in each class. Rabbit anti-mouse immunoglobulines were used as a
control. The data are consistent with our proposal that NIPP1 is associated with a large set of
Polycomb targets and these targets overlap with EZH2 bound loci.
In the new Fig. 7, we selected five genes that were trimethylated I H3K27, each from a
distinct group, as defined in Fig. 6. The RNAi-mediated knockdown of either NIPP1 or EZH2
resulted in a loss of trimethylation of three out of these five genes, consistent with the notion
that both NIPP1 and EZH2 are required for the initiation and/or maintenance of H3K27
trimethylation.
Minor points:
1. In Fig. 1 immunostaining with NIPP1-/- blastocysts showed the normal level of Ezh2 in
spite of the reduction of trimH3K27. The quality of panels for EZH2 staining is not good
enough and thus should be improved.
Reply:
The EZH2 stainings (Fig. 1a) were repeated and the figures are now of an excellent quality.
2. It should also be needed to state either female or male were used for each study, which
could potentially affect the subnuclear localization of EZH2 and trimethylated H3K27.
Reply:
4
Where appropriate we have indicated whether the used cells were male or female (indicated in
the cell culture section of materials and methods). In addition, we have now pointed out that
NIPP1, in contrast to EZH2, does not co-localise with the inactivated X-chromosome in the
female blastocysts outgrowths. (new Fig. S1 and new text on p5, second paragraph).