Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment

Thank you for sending your work entitled “Zinc finger protein Zfp335 is required for formation of the naïve T cell compartment” for consideration at eLife. Your article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), a member of our Board of Reviewing Editors, and 3 reviewers.

The referees’ comments, shown below, were both positive and constructive. After consultation, Michel Nussenzweig, our Reviewing editor, has decided that the conditional KO experiment suggested by referee #3 is not necessary.

Reviewer 1:

This work from Dr. Cyster’s group has identified Zfp335 as a novel regulator of naive T cell maturation. The authors have elegantly combined forward genetics, immune assays and transcriptional analysis, and the experiments were designed and performed well with convincing results presented. This is certainly an interesting study, but I have the following comments for the authors to address.

1) Although the mutant mice have reduced peripheral cells, the physiological consequences on immune responses are unclear. The authors should test this in models of T cell-dependent immune responses.

2) In Figure 3, the authors showed that thymic but not peripheral naive T cells have defects in the maintenance upon adoptive transfer. They then described defect in RTE as a likely mechanism, but have not directly tested the survival or maintenance of RTE cells. This needs to be done.

3) In Figure 4, the authors measured expression of Bcl2 family proteins to exclude a role of cell apoptosis. They should directly measure cell apoptosis, e.g. by caspase staining.

4) In Figure 5, the authors used ChIP-Seq on total thymocytes to identify Zfp335 targets. Since the most relevant cell type is mature thymocytes, they need to validate the target genes by performing ChIP experiment using mature thymocytes followed by qPCR of the targets.

Reviewer 2:

The manuscript by Han et al. describes the finding of a new Zinc finger protein Zfp335 in the development of mature thymocytes and peripheral T cells from the analysis of a mouse strain from ENU-mutagenesis. The developmental defects of T cells are restricted to the stages after DP thymocytes, particularly to mature SP, RTE, and peripheral T cells.

The causative gene was found to be a hypomorphic mutation of Zfp335 from the experiment by bone marrow reconstitution by retroviral transduction of the wild type and mutant Zfp335. By very extensive analyses using various TCR-Tg mouse models and systems, the authors suggested that the developmental defect could not be attributed to the impaired selection, cell survival, and thymic egress. To elucidate the defect by identifying the target genes of Zfp335, the authors found several possible target genes that Zfp335 directly binds. However, none of them could explain the developmental defect. One target gene Ankle2 may partly restore mature T cells.

Overall the analysis of the mutant mouse and gene demonstrated a critical role of Zfp335 in development of T cells and identify the target genes, but failed to neither reveal the mechanism of the developmental defect nor identify the function of Zfp335 gene.

1) Although Zfp335 restored mature T cell development by bone marrow reconstitution, it is recommended to confirm by the knock-in of Zfp335 because the mice may still contain other mutations and the heterogeneity of chimeric mice does not prove full restoration of development.

2) Thymic selection using OTII-Tg mice showed significant defect of development of mature TCR + T cells, the author might not simply neglect the effect on thymic selection.

3) The data for the effect of Ankle2 in the reconstitution experiment was complicated such as the analysis of Rag-GFPhigh and low population (for example why Thy1.1 expression levels were so different between Ankle2 vs. control). Simpler experiment and expression should be taken to show significant restoration from developmental arrest.

Reviewer 3:

In this manuscript, Han et al., describe the analysis of the T cell compartment in the mouse mutant bloto, carrying a hypomorphic mutation in the zinc finger protein gene Zfp335. The authors identify a missense mutation in Zfp335 that alters zinc finger 12 and affects the DNA-binding ability of the protein. Homozygous bloto mice show a defect in the formation of the naive T cell compartment that cannot be attributed to altered thymic selection, cell proliferation or survival. ChIP-seq and microarray analyses to identify Zfp335-occupied and -regulated genes indicate that a very small number of genes are differentially bound and regulated in mutant and wild type thymocytes. Overexpression of one of these targets, Ankle2, in Rag1-GFP+ naive T cells showed a partial rescue of the mutant phenotyope.

The extensive analysis of the T cell defect of Zfp335bloto mice and the molecular examination of the defect in DNA binding and gene transcription are interesting and extend previous work on the function of Zfp335 in neural stem cells (Cell 151, 1097, 2012). The data are convincing and well presented. In particular, the partial rescue of the mutant phenotype by the overexpression of one of the identified Zfp335 targets provides strong evidence for the functional role of this Zfp335-regulated gene. However, the study would gain additional significance by the analysis of mice carrying a conditional null allele of Zfp335. Such mice have been published, and ES cells that harbor a conditional null allele of Zfp335 are available from a mutant mouse repository.

1) The authors need to address and/or discuss the discrepancies between the previous and current studies of Zfp335. For example, in Figure 4S3 the authors show that cell proliferation and survival are not affected by the bloto mutation. However, the previous analysis of lymphoblastic cells of humans carrying a hypomorphic H111R mutation shows an impaired growth of mutant cells.

2) In the ChIP-seq analysis, shown in Figures 5 and 6, the authors used the same anti-Zfp335 antibodies as the previous study. However, the authors identified a different sequence motif than the previously reported. Which parameters were used in the MEME analysis? What are the other significantly enriched motifs? Did the analysis detect the motif described in the Cell paper? If the motif width range is adjusted, is the other motif detected? I appreciate the inclusion of an electrophoretic mobility shift assay, which provides strong evidence for the newly identified sequence motif. The authors should also include the previously reported motif in this analysis.

3) One of the strongest points of the paper is the partial rescue of the T cell defect in bloto mice by the overexpression of Ankle2 (Figure 8). It would be of interest to examine the phenotype of an Ankle2 knock-down.

Reviewer 1:

[…] This is certainly an interesting study, but I have the following comments for the authors to address.

1) Although the mutant mice have reduced peripheral cells, the physiological consequences on immune responses are unclear. The authors should test this in models of T cell-dependent immune responses.

We have tested the mice in different models of T cell-dependent immune responses, but found no effect. In the first model, OTII blt/blt T cells were able to expand and elicit an antigen-specific germinal center B cell response from lysozyme specific Hy10 BCR transgenic B cells following duck egg lysozyme (DEL)-OVA immunization, on par with OTII blt/+ controls. Similarly, blt/blt mice showed no significant impairment in a model of infection with PR8 influenza virus; they were able to mount a normal NP_366-374 tetramer-specific CD8⁺ T cell response despite having lower numbers of peripheral T cells. These findings may be thought of as consistent with our in vitro experiments showing that blt/blt naïve T cells proliferate normally in response to TCR stimulation (Figure 4–figure supplement 3C).

In summary, all the data we have accumulated up to this point suggest that the bloto T cell defect is mainly developmental and does not profoundly disrupt basic T cell function. Nevertheless, T lymphopenia negatively affects repertoire size and is generally associated with an increased risk of infection. It is likely that physiological consequences may be revealed when these mice are exposed to a broader range of infectious agents than are typically present in a barrier facility, or if they are infected with multiple pathogens simultaneously.

2) In Figure 3, the authors showed that thymic but not peripheral naive T cells have defects in the maintenance upon adoptive transfer. They then described defect in RTE as a likely mechanism, but have not directly tested the survival or maintenance of RTE cells. This needs to be done.

We thank the reviewer for raising this important point. To address this concern, we have performed additional adoptive transfer experiments of peripheral T cells using the Rag1-GFP reporter system, and found that blt/blt Rag1-GFP⁺ T cells underwent a steeper decline over time compared to control blt/+ Rag1-GFP⁺ T cells. These results (new Figure 3C, 3D) complement our analysis of thymic and peripheral T cells in the original Figure 3 (now moved to Figure 3–figure supplement 1B) and provide direct evidence for a defect in RTE maintenance. These data are discussed in the revised text.

3) In Figure 4, the authors measured expression of Bcl2 family proteins to exclude a role of cell apoptosis. They should directly measure cell apoptosis, e.g. by caspase staining.

We initially attempted annexin V and active caspase 3 staining in freshly isolated thymocytes and naïve T cells ex vivo, but our data did not reveal clear differences and were not included in our manuscript. It should be noted that the frequency of apoptotic cells detected was extremely low, likely because dying cells are rapidly and efficiently cleared in vivo, making it difficult to assess in vivo T cell death using these methods. We now make this point in the revised text.

In response to the reviewer’s comment, we have analyzed the survival of sort-purified mature CD4SP thymocytes following in vitro culture. blt/blt cells showed an increased rate of cell death (as determined by annexin V and DAPI staining) over time relative to co-cultured WT controls, whereas the same effect was not observed in blt/+ mature SP thymocytes (new Figure 4C). This suggests that blt/blt mature SP thymocytes have reduced viability, at least in vitro, which is likely to contribute to the defect in vivo. The failure of Bcl2 overexpression to rescue the peripheral T cell deficiency (Figure 4D) suggests the involvement of cell-death pathways other than those countered by Bcl2. We have revised the text to more clearly discuss this point.

4) In Figure 5, the authors used ChIP-Seq on total thymocytes to identify Zfp335 targets. Since the most relevant cell type is mature thymocytes, they need to validate the target genes by performing ChIP experiment using mature thymocytes followed by qPCR of the targets.

As requested, we have performed ChIP-qPCR on sort-purified CD4SP thymocytes and successfully validated the target genes in Figure 6, as well as reproduced the same pattern of differential Zfp335 binding at various targets shown in Figure 6D. These results have now been added as Figure 6–figure supplement 1A.

Reviewer 2:

[…] Overall the analysis of the mutant mouse and gene demonstrated a critical role of Zfp335 in development of T cells and identify the target genes, but failed to neither reveal the mechanism of the developmental defect nor identify the function of Zfp335 gene.

1) Although Zfp335 restored mature T cell development by bone marrow reconstitution, it is recommended to confirm by the knock-in of Zfp335 because the mice may still contain other mutations and the heterogeneity of chimeric mice does not prove full restoration of development.

While it is true that ENU mutagenesis gives rise to a scattering of mutations throughout the genome, we have mentioned in the text that our whole-exome sequencing analysis identified the Zfp335^R1092W mutation as the only novel homozygous single-nucleotide variant within the mapped interval of interest on chromosome 2, making it highly unlikely that the bloto phenotype is caused by some other mutation. Given this information and the amount of data we have supporting the causative role of the Zfp335^R1092W mutation, we hope that this reviewer will agree that generating a knock-in mouse is not critical in the context of the present study. In the longer term we agree that it will be valuable for comparisons to be made between the phenotype of Zfp335^R1092W mice and mice lacking Zfp335 selectively in T cells, and we make this point in the Discussion.

2) Thymic selection using OTII-Tg mice showed significant defect of development of mature TCR + T cells, the author might not simply neglect the effect on thymic selection.

As correctly pointed out and as stated in the text, OTII TCR-tg blt/blt mice had reduced numbers of Vα2⁺ CD4SP thymocytes. However, the magnitude of this decrease (approx. two-fold) was similar to what we see in non-TCR Tg blt/blt mice (Figure 4A vs. Figure 1D), which suggests that the defect in OTII mice probably represented the same maturation phenotype as that seen in polyclonal mice. If there were a thymic selection effect on top of the maturation defect, we would expect to see a greater fold reduction in OTII CD4SP thymocytes, which was not the case. However, we agree that we cannot rule out a possible influence of Zfp335^R1092W on thymocyte selection and we have revised the Discussion section in an effort to clarify this point.

3) The data for the effect of Ankle2 in the reconstitution experiment was complicated such as the analysis of Rag-GFPhigh and low population (for example why thy1.1 expression levels were so different between Ankle2 vs. control). Simpler experiment and expression should be taken to show significant restoration from developmental arrest.

The difference in Thy1.1 reporter levels between Ankle2 and control is one that we typically see in retroviral transduction experiments and may be explained by the fact that the control cells were transduced with an empty retroviral vector, which due to its smaller size compared to a vector containing a large gene like Ankle2 (2895 bp), is able to integrate into the host genome more efficiently and be present at higher copy numbers, resulting in a corresponding increase in reporter expression. We hope this additional explanation helps clarify what is admittedly a complex experiment as we are not aware of a simpler way to perform this analysis. We also note that the partial rescue effect of Ankle2 was not observed with five other Zfp335 target genes tested in reconstitution experiments (Figure 8–figure supplement 1A), involving a cumulative analysis of more than 30 retrovirally transduced blt/blt BM chimeric mice. We believe that this large comparison group adds strength to the conclusion regarding the small but significant pro-maturation effect of Ankle2.

Reviewer 3:

[…] The data are convincing and well presented. In particular, the partial rescue of the mutant phenotype by the overexpression of one of the identified Zfp335 targets provides strong evidence for the functional role of this Zfp335-regulated gene. However, the study would gain additional significance by the analysis of mice carrying a conditional null allele of Zfp335. Such mice have been published, and ES cells that harbor a conditional null allele of Zfp335 are available from a mutant mouse repository.

1) The authors need to address and/or discuss the discrepancies between the previous and current studies of Zfp335. For example, in Figure 4S3 the authors show that cell proliferation and survival are not affected by the bloto mutation. However, the previous analysis of lymphoblastic cells of humans carrying a hypomorphic H111R mutation shows an impaired growth of mutant cells.

We thank the reviewer for raising this point and have incorporated some of this discussion into the revised text. The H1111R mutation described in human patients by Yang et. al. was a far more severe hypomorph than the R1092W mutation present in our mice. The human mutation affected splicing, resulting in lower levels of normally spliced Zfp335 transcript and severely reduced protein expression in homozygous patient lymphoblastic cell lines (16% of control). In contrast, the R1092W mutation was comparatively benign: blt/blt cells have no decrease in Zfp335 expression at the transcript or protein level. A plausible reason for the discrepancy in proliferative capacity may be that H1111R mutant lymphoblastic cells express very little functional Zfp335, while blt/blt cells maintain sufficient Zfp335 activity such that they are capable of normal proliferation and growth. We now discuss this possibility in the text.

2) In the ChIP-seq analysis, shown in Figures 5 and 6, the authors used the same anti-Zfp335 antibodies as the previous study. However, the authors identified a different sequence motif than the previously reported. Which parameters were used in the MEME analysis? What are the other significantly enriched motifs? Did the analysis detect the motif described in the Cell paper? If the motif width range is adjusted, is the other motif detected? I appreciate the inclusion of an electrophoretic mobility shift assay, which provides strong evidence for the newly identified sequence motif. The authors should also include the previously reported motif in this analysis.

Using the set of parameters described in our Methods (min. width = 6, max. width = 30, zero or one instance of a given motif per sequence) for MEME analysis of our thymocyte ChIP-seq data, we did not detect the motif previously reported by Yang et. al., (Cell, 2012) within our top five highest ranked hits. Furthermore, analysis of our data using an alternative motif discovery algorithm, HOMER, did not reveal significant enrichment of this motif.

The MEME analysis parameters used by Yang et. al., differed from ours in two key aspects: 1) a maximum motif width of 20 (instead of 30) was specified; 2) discriminative motif discovery was performed using a negative set of “background” sequences contrasted against a set of target sequences extracted from the top 148 peaks in their ChIP-seq dataset.

Even after adjusting for motif width, our analysis yielded the same sequence motif that we identified, and not the previously reported motif. We have also re-analyzed the embryonic brain ChIP-seq data, extracting 400 bp sequences from the top 148 peaks identified by MACS, and running MEME with a maximum motif width of either 20 or 30. In both cases, our proposed motif emerged as the top-scoring candidate, whereas the motif reported by Yang et al., was not detected. Based on these analyses, we would argue that the discrepancy between our conclusions and that of Yang et. al., is likely not due to true differences in the underlying biological data; instead, differences in motif finding strategy, specifically with regards to the choice of a background model by Yang et. al., most likely account for this discrepancy.

As suggested by the reviewer, we have performed additional experiments to test the previously reported motif in an electrophoretic mobility shift assay (new Figure 7–figure supplement 1). The probe sequence was derived from the Zfp335 binding site at the promoter of Pdap1, a target gene identified by ChIP-seq in both embryonic brain and thymocyte ChIP-seq datasets. Our assay did not reveal evidence for Zfp335 binding to this motif in vitro: firstly, we were unable to detect formation of a gel-shift complex with labeled Pdap1 probe, and secondly, addition of excess unlabeled Pdap1 probe failed to compete against labeled Z1 probe (containing our identified motif) for binding to Zfp335. These new data provide further support for our newly identified Zfp335 recognition motif being a major target of Zfp335 binding.

3) One of the strongest points of the paper is the partial rescue of the T cell defect in bloto mice by the overexpression of Ankle2 (Figure 8). It would be of interest to examine the phenotype of an Ankle2 knock-down.

We agree, and in fact we had attempted to knock-down Ankle2 by transduction of bone marrow progenitors using two different shRNA constructs (selected as the best of three when tested in a cell line). Unfortunately, we were only able to achieve at most 50% knock-down of Ankle2 expression in naïve T cells as assessed by qRT-PCR. At that level of knock-down, there was no detectable effect on T cell maturation (unpublished data). In blt/blt T cells, Ankle2 expression is decreased by a far greater degree; at least ten-fold (Figure 8A), which suggests that our shRNA knock-down efficiency was insufficient to reveal any effects of Ankle2 on T cell development. In the future, we hope to attempt this experiment again using CRISPR to achieve more efficient knock-down.