SYNOPSIS: The goal of this project is to characterize the function of a newly identified zinc finger protein (ZNF206), which the applicant hypothesizes is a transcription factor that is important for inhibiting differentiation of human embryonic stem cells (hESCs). The applicant will use ectopic expression in hESC of tagged ZNF206 to identify targets and binding partners in genomic and proteomic studies. Similar overexpression and shRNA knockdown approaches will be used to assay its importance for differentiation/pluripotency using EB and teratoma assays.
IMPACT AND SIGNIFICANCE: This investigation is directed to the molecular mechanisms regulating early lineage commitment of hESCs to extra-embryonic primitive endoderm (PE) and is highly focused on a candidate molecular regulator of this early stage of lineage commitment. Knowledge of the particular factors that control these processes and modulate stem cell differentiation is important for designing better strategies to maintain these cells in culture and to direct their differentiation to particular cell lineages.
The investigators have preliminary data suggesting that PE may be a default pathway for inner cell mass-derived cells and believe this holds for hESCs as well. They further have data suggesting that PE may be pivotal for further lineage commitment for ICM-derived cells and suggest that allowing hESC to generate a clonally related PE will provide a culture condition favorable for lineage-specific differentiation to more mature cells such as neural and/or cardiac. The research has the potential to provide mechanistic information about early hESC differentiation and about how hESCs might be effectively manipulated to desired lineages which can importantly impact on the utilization of hESCs for a variety of therapeutic purposes.
The proposal is original in that it will focus on a new candidate hESC regulator (ZNF206), which the applicant has identified through transcriptional profiling of undifferentiated hESC vs. hESC differentiated toward the primitive endoderm lineage. ZNF206 co-localizes with the stemness genes OCT4 and NANOG and the expression of this novel gene product diminishes as hESC differentiate towards the primitive endoderm lineage, and it is not detected in adult tissues. The preliminary data suggests that ZNF06 may inhibit default differentiation towards extra-embryonic lineages, and the PI hypothesizes specifically that it may repress PE differentiation. Unfortunately, the evidence for ZNF206 involvement in ES function is limited to expression analysis, and thus remains circumstantial. The approaches, per say, involve primarily standard overexpression and knockdown assays, coupled with genomic and proteomic studies, and are not particularly innovative.
Given the focus on a single gene product, the potential impact of the proposal depends exclusively on the function of ZNF206. If ZNF206 mediates the switch from hESC to PE lineage, the proposed identification of transcriptional activity, binding partners, downstream mRNA and protein expression has the potential to generate exciting new information for how transcription factors control pluripotency of embryonic stem cells. These results could not only impact our understanding of the basic processes by which early embryonic cells commit to lineages, they could also apply to approaches of differentiating hESC towards particular target cell types. If ZNF206 does not affect hESC pluripotency, the impact will be considerably reduced.
The significance of the research lies in the extent to which it may enlighten us regarding the regulation of pluripotency and early lineage commitment of hESCs as well as in the understanding of the role of a potential new transcription factor.
QUALITY OF THE RESEARCH PLAN: The research plan involves standard expression profiling, molecular analysis, ChIP-on-Chip, proteomics, and over-/under-expression approaches to characterize the importance of ZNF206 in ES cells. While the overall quality of the research plan is good, what is most obviously lacking in this proposal is preliminary data demonstrating that this factor does indeed play a role in hESC differentiation. In addition, the proposal lacks important detail regarding several aspects of the methods/data. First, how was ZNF206 chosen as the primary candidate for further testing? How many other factors showed statistically similar regulation of expression? The data arguing for selective expression of this factor in ES is limited in that the comparison was done only to whole tissue, and so expression in rare cells (perhaps tissue stem cells) would not be observed by this approach.
The research plan focuses on two aims. The first is to demonstrate that ZNF206 is a transcription factor and to determine its characteristics & binding partners in hESCs through a combination of biochemical & cell biological approaches. The second aim is to determine the functional significance of ZNF206 by testing by silencing or overexpression experiments whether it regulates hESC self-renewal and pluripotency through its inhibition of PE differentiation as hypothesized.
The first aim will be explored via biochemical and cell biological approaches that ask where ZNF206 localizes in hESCs, test its association with specific co-factors or DNA sequences and whether it has preferred DNA binding specificity. Expression of several tagged versions of ZNF206 will be introduced to determine intracellular localization (EGFP and V5 tags), interacting proteins (TAP tag), and DNA binding sites (V5). In addition, purified recombinant protein from bacteria will be used to determine DNA binding specificity of ZNF206.
Each of the approaches involving hESC clones is described well and is likely to produce useful information regarding the molecular characteristics of ZNF206. The feasibility of the experiments is supported by: 1) Dr. Snyder’s group has already generated the necessary lentiviral vectors and tested them in cells. 2) They have demonstrated an efficient method of generating clonal lines of transduced hESC, which will allow them to select from clones expressing optimal levels of tagged ZNF206 for each approach. 3) They have established a collaboration with Dr. John Yates to perform MudPIT analysis to identify interacting proteins. Regardless of the effects of ZNF206 expression on hESC characteristics, the approach here will generate a better understanding how ZNF206 performs its function.
For the second aim, lentiviral-mediated overexpression and knockdown or silencing experiments will be performed to modify ZNF206 function in hESCs. Clones will be generated and the effects of altered expression levels will be examined by clonality and pluripotency assays to test for self-renewal, differentiation and pluripotency. Downstream targets (RNA and protein) affected by altered ZNF206 expression will be determined by genomic and proteomic approaches. The experiments are straight-forward, carefully described and the methods are all within the investigators armamentarium. These experiments will determine if ZNF206 is important for hESC self-renewal.
The reviewers had various degrees of concern with the design of the research if ZNF206 IS vital for hESC pluripotency. A reviewer noted that in this circumstance the PI may have significant difficulty with the over and under-expression approaches proposed, as these may be incompatible with maintenance of hESC, and conditional expression approaches are not described. This is a problem also for the use of ectopically expressed tagged protein in ChIP-on-chip and proteomic studies. How will expression levels be controlled, as different levels of expression may yield different targets or binding partners? To really analyze physiological targets/partner, it may be necessary to raise new antibodies against the protein.
Another reviewer cited as “one interesting possible pitfall” for the forced expression of ZNF206 deals with the evidence that differentiation into PE aided the recovery of clonal hESC cultures in a defined, feeder free culture. If ZNF206 inhibits PE lineage commitment, and the formation of PE derivative from hESC colonies supplies paracrine factors stimulating hESC self-renewal, then forced expression of ZNF206 could actually inhibit hESC self renewal in feeder-free cultures. Clonality assays may need to be performed on inactivated PEL cells or PEL-conditioned media. Although this is only a minor concern, it could introduce an undesirable extra layer of complexity
STRENGTHS: The primary strength of the proposal lies in its focus on a circumscribed set of questions that provides potential for important discoveries relating to a putative transcription factor that may be a novel candidate regulator of pluripotency and differentiation. The knowledge obtained regarding ZNF206, a previously uncharacterized gene product, will be valuable, although it is understood that the outcome may be a negative one, rather than the positive association the investigators propose. The investigators anticipate a variety of pitfalls and propose reasonable alternative approaches in the event these occur. The research offers potential for novel discoveries of molecular genetic determinants of hESC self renewal.
The research team is a talented group of investigators led by an established investigator.
WEAKNESSES: The obvious weakness of this application is in the possibility that the very circumscribed focus and limited preliminary data regarding ZNF206 may result in the entire approach turning out to be a blind pathway. The reliance on analysis of a single gene product without any data regarding its function is risky. Although expression data supports a potential role for ZNF206 in preventing PE differentiation of hESC, if these changes are merely coincidental, then the impetus for identifying its target and co-factors would be significantly reduced and it is entirely possible that perturbation of ZNF206 expression could have no effect on hESC self renewal or differentiation. This risk has to be weighed in light of the potential value of the information that is obtained. Given that so little is known of the early pathways regulating hESCs and determining their lineages, the risk of this narrow approach might be justified but additional preliminary data demonstrating importance of ZNF206 for ESC function would have greatly strengthened the proposal and provided greater assurance of value.
Other weaknesses included the lack of discussion of how this protein was chosen as particularly interesting; a lack of demonstration of efficacy of overexpression/knockdown approaches in hESC (most data is with 293T or Hela) and concern about experimental design for proteomic and genomic studies (overexpression of tagged protein)
DISCUSSION: This application proposes a circumscribed set of questions to characterize and determine the role of a novel protein identified by transcriptional profiling that is a putative transcription factor and is hypothesized to be a regulator of hESC pluipotency and early differentiation. The biggest concern of all the reviewers was that the preliminary data was viewed as indirect and/or insufficient to support the proposed hypothesis of this factor as a regulator of hESC pluripotency and early differentiation. The proposed research was therefore regarded as a gamble. As one reviewer noted, the proposal suffers from the Samson phenomenon, its strength is its weakness. The factor is everything; if they’re wrong, there’s nothing or at least no information that is likely to be valuable in the same way and there are no alternative plans to address this situation. The reviewers’ noted that a little preliminary data that perturbation of this factor affects function would have increased their enthusiasm for this proposal
There was also discussion as to the rationale for choosing this protein from the transcriptional profiling; again what was provided was viewed as insufficient. There was also the comment that it has been proven not to be the case that endoderm is the default differentiation pathway.