Zinc finger protein Zfp335 controls early T-cell development and survival through β-selection-dependent and -independent mechanisms
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Evaluation Summary:
The authors have discovered that the transcription factor Zfp335 is an important regulator of early T cell development in the thymus. This paper will be of interest to scientists within the field of T cell development. The approaches used are thoughtful and rigorous and the key claims are supported by the data. Whether or not Zfp335 specifically controls beta-selection via the gene targets described requires additional experimentation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
T-cell development in the thymus undergoes the process of differentiation, selective proliferation, and survival from CD4 − CD8 − double negative (DN) stage to CD4 + CD8 + double positive (DP) stage prior to the formation of CD4 + helper and CD8 + cytolytic T cells ready for circulation. Each developmental stage is tightly regulated by sequentially operating molecular networks, of which only limited numbers of transcription regulators have been deciphered. Here, we identified Zfp335 transcription factor as a new player in the regulatory network controlling thymocyte development in mice. We demonstrate that Zfp335 intrinsically controls DN to DP transition, as T-cell-specific deficiency in Zfp335 leads to a substantial accumulation of DN3 along with reduction of DP, CD4 + , and CD8 + thymocytes. This developmental blockade at DN stage results from the impaired intracellular TCRβ (iTCRβ) expression as well as increased susceptibility to apoptosis in thymocytes. Transcriptomic and ChIP-seq analyses revealed a direct regulation of transcription factors Bcl6 and Rorc by Zfp335. Importantly, enhanced expression of TCRβ and Bcl6/Rorc restores the developmental defect during DN3 to DN4 transition and improves thymocytes survival, respectively. These findings identify a critical role of Zfp335 in controlling T-cell development by maintaining iTCRβ expression-mediated β-selection and independently activating cell survival signaling.
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Evaluation Summary:
The authors have discovered that the transcription factor Zfp335 is an important regulator of early T cell development in the thymus. This paper will be of interest to scientists within the field of T cell development. The approaches used are thoughtful and rigorous and the key claims are supported by the data. Whether or not Zfp335 specifically controls beta-selection via the gene targets described requires additional experimentation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors identified a new regulatory pathway involved in T cell development centred on a transcription factor called Zfp335. They demonstrate that Zfp335 deficiency resulted in reduced development, in vivo and in vitro, due to blockade in development at the DN3 stage. This was associated with reduced intracellular TCRβ and increased apoptosis. The authors undertook transcriptomic and ChIP-seq analyses and determined that Zfp335 interacts with Bcl6 and Rorc and retroviral expression of either of these transcription factors countered the effects of Zfp335 deficiency.
Strengths
The paper is easy to understand and the results are presented in clear and logical manner. The authors identify a novel transcription factor that had a clear impact on T cell development, which is exciting as it helps expand our …Reviewer #1 (Public Review):
The authors identified a new regulatory pathway involved in T cell development centred on a transcription factor called Zfp335. They demonstrate that Zfp335 deficiency resulted in reduced development, in vivo and in vitro, due to blockade in development at the DN3 stage. This was associated with reduced intracellular TCRβ and increased apoptosis. The authors undertook transcriptomic and ChIP-seq analyses and determined that Zfp335 interacts with Bcl6 and Rorc and retroviral expression of either of these transcription factors countered the effects of Zfp335 deficiency.
Strengths
The paper is easy to understand and the results are presented in clear and logical manner. The authors identify a novel transcription factor that had a clear impact on T cell development, which is exciting as it helps expand our understanding of this important process.Weaknesses
I felt a weakness of the paper was that the authors identified a number of processes that were impacted by this transcription factor but did not look at the interaction between them. Furthermore, the discussion would benefit from more discussion of previous research to put the results into context, including other regulatory pathways involved in T cell development. -
Reviewer #2 (Public Review):
In this paper the authors address the role of the Zinc finger protein Zfp335 in early T cell development. Earlier studies established that a Zfp335 hypomorph mutant mouse had defects in T cell development. Here the authors show that conditional ablation of Zfp335 using LckCre results in a dramatic reduction of thymic cellularity and a developmental block at the DN3 stage of development. Using OP9-DL1 cultures and mixed BM chimeras they establish that the developmental defect is cell intrinsic. Here it is not clear why the authors use a starting ratio of 1 to 4 WT to KO and whether they take into account this starting difference in the final calculations. Curiously LckCre mediated ablation of Zfp335 also impacts gdT cell development which branches independently of the preTCR assembly and signaling. Then the …
Reviewer #2 (Public Review):
In this paper the authors address the role of the Zinc finger protein Zfp335 in early T cell development. Earlier studies established that a Zfp335 hypomorph mutant mouse had defects in T cell development. Here the authors show that conditional ablation of Zfp335 using LckCre results in a dramatic reduction of thymic cellularity and a developmental block at the DN3 stage of development. Using OP9-DL1 cultures and mixed BM chimeras they establish that the developmental defect is cell intrinsic. Here it is not clear why the authors use a starting ratio of 1 to 4 WT to KO and whether they take into account this starting difference in the final calculations. Curiously LckCre mediated ablation of Zfp335 also impacts gdT cell development which branches independently of the preTCR assembly and signaling. Then the authors suggest that the developmental bock is in the transition from the DN3 to DN4 stage which is not surprising given that LckCre mainly targets DN3 cells. The authors do not provide further detail on the transition between the DN3a and DN3b stages. The knockout cells do not seem to have a defect in proliferation however they have increased apoptosis both at the DN3 and at the DN4 stage. The authors then show that a reduced fraction of the mutant DN4 cells expresses intracellular TCRb and that providing OT1, a mature abTCR, to the developing cells resolves the transition to the DN4 stage but not to the DP.
The authors then provide molecular analyses Using RNA seq in WT and KO DN4 cells they identified 566 downregulated, and 899 upregulated genes. They specifically focus only on the downregulated genes and show a heatmap with a number of hand peaked downregulated genes with roles in thymocyte development. They also perform ChIPseq of Zfp335 in FACS sorted mix of DN3 and DN4 WT thymocytes indicating that they isolated 5x107 cells of these rear cell populations for the ChIP experiment. They identify 2797 peaks. It is not clear how many independent ChIP-seq replicas were processed neither is it clear whether the identified peaks are enriched for the consensus Zfp335 binding motif as a means of validating the ChIP. The authors also indicate a GEO accession number that could answer these questions but access to this site is still blocked. Then the authors compare a select number of 97+22 differentially expressed genes and it is not clear how these genes were peaked from the 566 downregulated and 899 upregulated genes identified as differentially expressed. Using this methodology the authors narrow down to Bcl6 and Rorc which are downregulated and are marked by Zfp335 binding at TSS proximal regions of Bcl6 and Rorc.
The authors then perform functional assays in which they retrovirally overexpress Mock, Zfp335, Bcl6 or Rorc in KO DN3 thymocytes cultured on OP9-DL1 that results in a certain rescue of development and reduction in apoptosis. They have not assessed whether a similar retroviral overexpression of Zfp335, Bcl6 or Rorc in WT DN3 cells could have affected the progression of these cells.
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Reviewer #3 (Public Review):
Wang et al. report the identification of Zfp335 as a novel regulator of T cell development and demonstrate that it is critical for the DN to DP transition. Their conclusions are supported by the analysis of Zfp335 deficient mice, by careful phenotyping of the blocked. By employing adoptive transfer experiments in vivo and OP-DL1 cultures in vitro, they confirm that the requirement of Zfp335 is intrinsic to developing thymocytes. A strength of the study was the application of ChIP analysis to identify targets of Zfp335 and validating the function of two targets, Bcl6 and Rorc, by transduction into Zfp335 deficient cells to rescue the developmental block. Overall, this is an interesting study and reveals new knowledge about the regulation of the DN to DP transition in T cell development.
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