Hic2 fast-tracks iPS cell generation by suppressing KLF4-dependent epidermal detour

  1. Meryam Beniazza
  2. Masahito Yoshihara
  3. Daniel F Kaemena
  4. James Ashmore
  5. Suling Zhao
  6. Michael O’Dwyer
  7. Emil Andersson
  8. Victor Olariu
  9. Shintaro Katayama
  10. Abdenour Soufi
  11. Kosuke Yusa
  12. Keisuke Kaji

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Abstract

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is the most established cellular conversion by exogenous master transcription factors (TFs). A deeper understanding of this yet inefficient process is critical to extending our capability to control cellular identity for medical applications. Here we report 14 genes essential for efficient iPSC generation, but dispensable for self-renewal. Of those, overexpression of Hic2 , a transcriptional suppressor highly expressed in PSCs, enhances iPSC generation ∼10-fold. This is achieved through a more direct transition towards pluripotency, bypassing an intermediate state with KLF4-dependent transient epidermal gene expression during iPSC generation. Mechanistically, HIC2 co-occupies these KLF4 targets and directly inhibits their expression. Our work demonstrates that master TFs necessary for cellular conversions can also activate obstructive genes during cellular reprogramming. We propose that identifying transcriptional suppressors against such side effects, like Hic2 , can be a powerful strategy to achieve more efficient TF-mediated cell conversions.

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  1. Review Commons

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    I thank the Referees for their...

    Referee #1

    1. The authors should provide more information when...

    Responses

    • The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A).
    • Though this is not stated in the MS
    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

    Response: We added...

    Referee #2

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This is a good manuscript, well performed and well presented. I have several suggestions/questions to enhance the clarity of the concept, as technically the work is rather well performed.

    1. I suggest that the authors explain better the mesenchymal-to-epithelial (MET) transition in reprogramming. Perhaps, explaining that epithelial gene acquisition (e.g., CDH1) and epidermal cell fate are not exactly the same. This approach could also be used to divide the genes they study further in their analyses.
    2. KLF4 is both a repressor and an activator in different cell contexts including reprogramming. Does HIC2 act only as repressor? Is it possible that HIC2 is repressing KLF4-activated genes bad for reprogramming (including epidermal genes) and activating KLF4-suppressed genes ncessary for reprogramming? This should not be too difficult to explore with their current dataset and they also could look at available datasets for histone modifications in reprogramming.
    3. Does HIC2 bind to genes related to somatic cell identify that need to be suppressed in reprogramming before the MET phase takes place?
    4. Does HIC2 influence proliferation during reprogramming?

    Referee cross-commenting

    Comments by the other reviewers are sound and will help improve the manuscript.

    Significance

    In this manuscript, Kaji and colleagues perform a CRISPR/Cas9 screen to identify genes involved in mouse somatic cell reprogramming, identifying HIC2 as a target that they further validate. They conclude that HIC2 acts by repressing the epidermal/epithelial program induced by KLF4 during reprogramming. Studying the complex role of transcription factor interactions in the context of cell fate conversions (of any kind and not just somatic cell reprogramming) is highly relevant. This work helps clarify such complexity in a specific context but the work has wider conceptual implications.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The study by Beniazza et al. aims to address the inefficiencies associated with OSKM-mediated reprogramming. Through a genome-wide CRISPR/Cas9 knockout screen, the authors identified 14 genes essential for iPSC reprogramming but dispensable for ESC self-renewal. Among these, HIC2 significantly enhanced reprogramming efficiency, yielding approximately a tenfold increase compared to standard conditions. scRNA-seq analyses revealed that HIC2-overexpressing cells follow a more direct trajectory toward pluripotency, bypassing the KLF4-dependent activation of keratinocyte and epidermal gene programs. ChIP-seq profiling further demonstrated that HIC2 and KLF4 co-occupy approximately 60% of their genomic targets, indicating substantial regulatory overlap. Notably, this co-binding and its functional effects are dose-dependent on KLF4, as shown by experiments comparing high KLF4 expression systems (standard OSKM and STEMCCA+9 constructs) with low KLF4 conditions (STEMCCA cassette lacking additional KLF4). The authors conclude that HIC2's modulatory effect occurs specifically under high KLF4 levels.

    Major Comments

    Figure 1D: What is the efficiency of gRNA library transduction into MEFs? What percentage of MEF cells were successfully knocked out? Figures 2B/C: To rule out the possibility that the observed variability in reprogramming efficiency among the tested factor combinations stems from differences in MKOS expression levels, the authors should provide evidence showing that the expression levels of all MKOS factors are comparable across samples. Figures 2D/E: To rule out a fibroblast-specific effect, can the authors show whether the epidermal gene signature is also upregulated during NSC reprogramming and whether Hic2 overexpression suppresses this signature? Figure 2H: Are the 13 signature genes that distinguish MKOS-Hic2-iPSCs from MKOS-iPSCs consistently identified across independent Hic2-iPSC lines, or does each reprogramming event produce a distinct gene set? If the signature is consistent, this is an important observation and should be further addressed and discussed. Figure 3K: Can the authors show the expression levels of MKOS and Hic2 transgenes in all samples? The same concern applies to Figure 4I. The reviewer wishes to be confident that the reduction in epidermal gene expression observed in MEFs is not due to variable transgene expression caused by multiple vector introductions (e.g., KLF4 alone versus KLF4 + Hic2), which could potentially lead to lower KLF4 expression through co-transfection competition. Does KLF4 overexpression in Hic2-knockdown MEFs lead to greater upregulation of the epidermal gene signature compared to the wild-type control? Figure 4C: It appears that only about half of the Hic2 binding sites overlap with KLF4 sites. What are the characteristics of the other Hic2-specific sites, and how might they contribute to reprogramming, if at all? Can the authors perform a reprogramming experiment using a combination that lacks KLF4 (e.g., replacing KLF4 with Esrrb or BMP4, as shown in PMID: 19136965 and PMID: 21135873) and test the effect of Hic2 under these conditions? Do KLF4 and HIC2 physically interact? The authors should perform a co-immunoprecipitation assay to address this question. What is the effect of Hic2 during human reprogramming? Does it play a similar regulatory role?

    Minor Comments

    • Typographical errors should be checked and avoided; for example, on page 10, the word 'colonies' was misspelled.
    • Some blank squares appear in the Methods section; please correct these formatting errors.

    Referee cross-commenting

    All suggestions are feasible within a relatively short time frame and will improve the manuscript.

    Significance

    Overall, this study is of significant interest to the stem cell community and presents a well-designed and carefully executed experimental framework. However, several concerns remain that should be addressed prior to publication.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: This work identified 14 genes essential for iPSC reprogramming but not essential for ESC maintenance and MEF proliferation by analyzing three CRISPR/Cas9-mediated genome-wide KO screens. Among them, they found that overexpression of the Hic2 gene can greatly promote OSKM-driven reprogramming. By using scRNA-seq in time points of the reprogramming process, they found that Hic2 can bypass the epidermal gene expressing state during reprogramming. Then, using ChIP-seq, they found that HIC2 and KLF4 have common binding sites on epidermal genes. Finally, by expressing KLF4 alone or KLF4 and HIC2 together, they demonstrated that HIC2 can inhibit KLF4-driven epidermal gene expression.

    Major comments: The claims and conclusions are well-supported by the data and do not require additional experiments or analysis. The data and methods are presented in a reproducible way.

    Minor comments: There seem to be some typos. For example, "we selected 30 genes with low FDRs in ESC maintenance" may be "high depletion FDR," since you want nonessential genes for ESC maintenance. The "log10(-FDR)" may be "-log10(FDR)." Some figures lack P values. Perhaps it would be useful to analyze whether Hic2 reduces reprogramming heterogeneity. Validation experiments, such as trilineage differentiation, could be considered to demonstrate that Hic2 does not affect the pluripotency and differentiation capacity of iPSCs.

    Significance

    General Assessment: This work is based on three CRISPR/Cas9-mediated genome-wide KO screens, which makes it comprehensive and reliable. They discovered that HIC2 and OSKM can drive reprogramming without an epidermal gene expression intermediate. They also found extensive common binding sites of HIC2 and KLF4 at target genes. This work not only enables more efficient reprogramming but also expands our understanding of the reprogramming process. Among HIC2 and KLF4 common target genes, some are repressed while others are activated, and it will be very interesting to study the mechanism of this selective function.

    Advance: Compared to natural `embryonic development, OSKM-driven reprogramming is very inefficient, and our understanding of the mechanisms of efficient reprogramming remains poor. The specific role of the epidermal gene expression state in the reprogramming process remains unclear. This work strongly supports the idea that repression of the epidermal gene expression state can promote iPSC generation. Moreover, previous studies on Hic2 are limited, and this work enriches our understanding of its mechanisms and functions.

    Audience: This study may be of interest to those interested in basic research on reprogramming mechanisms or Hic2, as well as those developing efficient reprogramming technologies.

    My field: Reprogramming, stem cells, aging, transcription factors.

  5. Version published to 10.1101/2025.09.11.675597 on bioRxiv