The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression

Thank you for sending your manuscript titled “ZNF644 and WIZ regulate the G9a/GLP complex for gene repression” for consideration for publication in eLife. Your manuscript was reviewed by three experts in the field and by a member of the Board of Reviewing Editors (BRE). After a full discussion of the study and the reviews, we are happy to report that the reviewers and the BRE member found the study of interest to the journal and therefore, we will be pleased to consider a revised manuscript addressing the following issues:

1) Provide statistical analyses and significance for the quantitative PCR studies and for the data on changes in gene expression when the G9a-GLP complex components are depleted.

2) Provide comprehensive genome-wide analysis including metagene, whole gene distributions, and average promoter analysis for the observations made in the manuscript.

3) Provide experimental evidence for direct interactions of ZNF644 and WIZ to the DNA sequence studied in the manuscript.

4) Provide a deeper discussion between the current analysis and the previous studies in terms of domains of interaction between WIZ and G9a-GLP. Also clarification on the difference between Feinberg's studies on the requirement of megabase size domains of histone H3K9me2 Vs the current study on the role of promoters in this process.

5) The manuscript needs to be read carefully for grammar and clarity.

Further details can be found in the comments below.

Reviewer #1

Significance: It was previously known that WIZ associates with and stabilizes the G9a-GLP heterodimer, but this paper identifies a new binding partner ZNF644. Furthermore, in a major breakthrough the paper demonstrates that WIZ and ZNF644 bind directly to specific DNA sequences and thereby target G9a to specific chromosomal locations. Previously, it was known that a few repressive DNA binding transcription factors can recruit G9a to specific chromosomal loci, but this paper describes a major new mechanism for targeting G9a and GLP to chromatin. The scientific quality of the data presented is generally quite good, but with a few exceptions noted below. There are significant English problems with the manuscript.

Major concerns: The quantitative PCR data for ChIP and for RNA expression throughout Figures 4–6 (including the supplements) lack any statistical analysis. The meaning of the error bars is not indicated as well as whether they represent technical or biological replicates and how many. Reproducibility in multiple biological replicates is not discussed. In many cases in these figures the error bars suggest that the differences seen and described in the text may not be significant. In particular, the changes in target gene RNA levels caused by depleting components of the G9a-GLP complex are quite small and appear to be of questionable statistical significance.

Overall recommendation: This is an exciting paper because it reports a new mechanism for targeting of G9a-GLP to specific chromosomal locations. Statistical analyses need to be provided for the quantitative PCR data. Furthermore, the data on changes in gene expression when G9a-GLP complex components are depleted is weak and of questionable statistical significance. This data should either be improved (at least to statistical significance) or eliminated. While it would be nice to have an indication that G9a-GLP is having an effect on gene expression at the sites where they are recruited, the novel targeting mechanism would still, in my opinion, be important enough to merit publication. English needs to be improved throughout the manuscript.

Reviewer #2

G9a-GLP is important for assembly of H3K9 dimethylated facultative heterochromatin. A major question is how does the complex localize to its sites of action? This is a key question in the field of chromatin biology. The authors show that the zinc finger TFs WIZ and ZN644 associate with G9a-GLP in vivo. They map interacting domains by co-IP with deletion mutants and present genome wide ChIP data on co-localization of the complex and locus-specific ChIP in various RNAi mutants. The interaction with WIZ was previously shown so the novelty here is the association with ZN644, the identification of binding motifs and validation of co-localization genome wide. These are important findings of broad interest in the field.

The major drawbacks are the lack of a comprehensive genome wide analysis (i.e., metagene, whole gene distributions, average promoter analysis and so on). Additionally, while the authors show strong evidence for targeting of G9a-GLP by WIZ and ZNF644, this is not linked globally with H3K9 dimethylation. There is an overemphasis on locus-specific ChIP studies that lacked measures of statistical significance. Moreover, the paper lacked a deeper discussion of and comparison with other discoveries in the field. For example, there is some disagreement between the authors' analysis and previous studies in terms of domains of interaction between WIZ and G9a-GLP. Additionally, Feinberg showed megabase size domains of histone H3K9me2, while the authors' studies focus on the promoters. Is WIZ-ZNF644-G9a-GLP binding inside as well as at gene promoters? Finally, there are a huge number of disappointing grammatical issues.

In general, I felt that the paper was a bit underdeveloped throughout but has the potential to make a significant contribution.

Reviewer #3

The manuscript from Bian and colleagues describes the interaction of G9A/GLP heterodimeric methyltransferase complex with two zinc finger containing proteins, Wiz and ZNF644. G9A/GLP activity results in heterochromatin formation and gene silencing. Affinity purification of G9A and ZNF644 was used to determine that G9A/GLP, Wiz and ZNF644 form a core complex. Wiz was shown previously to associate with G9A/GLP and alter its stability (Udea et al.); however, ZNF644 is a new component identified by this manuscript. Using immunoprecipitations of deletion mutants the authors determine that the TAD Domain of GLP and G9A interact selectively with WIZ and ZNF644, respectively. The authors go on to identify the genomic location of WIZ and ZNF644 and that the sites occupied by these proteins accounts for the majority of G9A occupied sites in the genome. Recruitment of G9A and the downstream readers of H3K9 methylation are effected by suppressing WIZ expression. Overall, the data make a compelling case for an important role of WIZ and ZNF644 in recruiting G9A/GLP to their genomic locations. To fully support the proposed role of these proteins in recognition of select genes through a defined DNA sequence, the following points should be addressed:

1) This implication of the requirement of ZNF644 or WIZ for GLP/G9A recruitment is that the zinc-finger domains of these proteins mediate sequence specific DNA interactions. Although the authors identify motifs within genes that are bound by the GLP/G9A/WIZ/ZNF644 complex, the ability of ZNF644 and WIZ to directly bind these DNA sequences is not addressed. The authors should determine if WIZ or ZNF644 selectively and directly recognize the motifs identified in Figure 4 by gel shift or similar assay.

2) The authors identify deletion mutants in ZNF644 and WIZ that fail to bind GLP and G9A. They go on to use these mutants in Figure 3 and 6 to suggest eliminating the ability of GLP or G9A to bind the zinc finger proteins results in an inability of the methyltransferases to stably associate with DNA and suppress transcription. However, for the data in Figures 3 and 6 to fully support the author's hypothesis the D1 and D8 mutants must be shown to stably interact with the DNA: 1) by selective extraction (Figure 3A) and 2) by ChIP. The trivial explanation for the existing data is that these mutants themselves fail to be recruited to DNA.