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In response to Li et al.: Linker histones function in Drosophila embryogenesis

Albert Carbonell, Lazslo Henn, Juan Pérez-Roldán, Srividya Tamirisa, Anikó Szabó, Imre M. Boros, Fernando Azorín

Preprint posted on 24 March 2020 https://www.biorxiv.org/content/10.1101/2020.03.21.001529v1

When your results are challenged, revise and report the updated findings.

Selected by Sree Rama Chaitanya

Context

Homodimers of four histones (H2A, H2A, H3, H4) form an octamer protein complex to wrap ~146bp DNA to form the building blocks of chromatin – nucleosome. Transcription, replication, and histone post-translational modifications (referred to as ‘histone code’) maintain a two-way relation to orchestrate the genome function. However, an underappreciated linker histone H1 binds at the DNA exit/entry gate of the nucleosome to facilitate high-order chromatin structure in turn to regulate a plethora of gene regulatory processes1. The presence of multiple somatic and germline-specific H1 variants in metazoans, make it challenging to study H1 biology. But thanks to Drosophila, that harbors a single somatic H1 variant (dH1) and a single germline-specific H1 variant (dBigH1), promising as a tractable organism to unravel the complete biology of H12.

In the previous work3, the host lab identified the germline-specific dBigH1 in the Drosophila embryos that regulate zygotic-genome activation (ZGA); dBigH1 is eventually replaced by somatic dH1 at the onset of cellularization. Moreover, they also demonstrated loss-of-function dBigH1 mutation (bigH1100) exhibits a higher lethality rate during embryogenesis. But challenging this finding, another group posted a preprint (Li et al. 2019) reporting that complete loss of dBigH1 does not induce embryonic lethality, with dH1 compensating the loss4. Therefore, the host lab of the initial study3 set forward to investigate this further and revisit their earlier findings.

Key outcomes

In the current preprint, the authors generated two null dbigH1 alleles – bigH1NULL and bigH1STOP – using CRISPR. In bigH1NULL, the complete coding sequence is replaced by mCherry, and in bigH1NSTOP, a stop codon was introduced at the N-terminal domain. dBigH1 was not detectable in ovaries and early embryos in either of these mutants. Intriguingly, they did not observe a significant effect on the embryo viability, supporting the claims in the Li’s preprint4. Not just that, the somatic dH1 levels were augmented in homozygous bigH1NULL and bigH1NSTOP mutants, but not in their earlier mutant bigH1100 carrying flies3 (Fig. 1). They suggest that a lack of compensatory dH1 takeover could be the cause of incomplete loss of dBigH1 in bigH1100 mutant flies. The authors’ further reasoned that acquired secondary mutations could have contributed to embryonic lethality in bigH1100 flies.

The current preprint took a positive approach to unravel the discrepancies in results raised by another group. All in all – this is a great reminder to revisit and challenge our findings and importantly report the revised results to the scientific community.

Fig. 1: Compensatory dH1 expression. A) Immunofluorescence staining with a dH1 antibodies (in green) of wild-type Drosophila embryos at the cellular blastoderm stage (left) and before nc 7 (right).  As in (A), embryos collected from bigH1100/TM6B parents (B),  homozygous bigH1NSTOP parents (C), and homozygous bigH1NULL parents (D). DNA was stained with DAPI (in red). Insets show enlarged images. Taken directly from Fig. 2 of Carbonell A et. al., 2020 under a CC-BY 4.0 international license.
Acknowledgments

I am grateful to all the authors for their support, especially Dr. Fernando Azorin for being open to discuss the unpublished results and replying promptly.

References:

  1. Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26.
  2. Bayona-Feliu A et. al., Histone H1: Lessons from Drosophila. Biochim Biophys Acta. 2016 Mar;1859(3):526-32.
  3. Pérez-Montero S et. al., The embryonic linker histone H1 variant of Drosophila, dBigH1, regulates zygotic genome activation. Dev Cell. 2013 Sep 30;26(6):578-90.
  4. Kaili K. Li et. al., Compensatory replacement of the BigH1 variant histone by canonical H1 supports normal embryonic development in Drosophila. bioRxiv 789735; doi: https://doi.org/10.1101/789735
  5. Henn L et. al., Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo. Nucleic Acids Res. 2020 Sep 18;48(16):9007-9018.
  6. Kosicki M et. al., Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018 Sep;36(8):765-771.

Tags: drosophila, linker histone h1, zga

Posted on: 29 September 2020

doi: https://doi.org/10.1242/prelights.24984

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Author's response

Dr Fernando Azorin (FA) shared

1) Your work is laudable and inspiring to any scientist on how to take criticism positively and set forth an example to report revised findings, however small it may be. Do you have any comments? It will be interesting to hear your side of the story.

FA: Certainly, we were intrigued by the findings reported by Li and collaborators from Yikang S Rong’s lab4. However, to some extent, their findings were not unexpected since, in late 2018, Laszlo Henn from Imre Boros’ lab in Szeged shared with us some very interesting observations, which have been recently published5. Laszlo generated CRISPR/CAS9 lines in which the CDS of dBigH1 was replaced by that of somatic dH1. These lines, though showing some embryo defects, were viable. At that point, it became clear that somatic dH1 was largely rescuing dBigH1 loss-of-function (LOF), raising some doubts on the actual dBigH1 function in embryogenesis. Thus, we started a collaboration to address these questions and, as a first step, we decided to generate new dBigH1 LOF alleles by CRISPR/CAS9. We were in the middle of that when Li’s report came out. From this point of view, Li’s work4 was very enlightening and we were happy to reproduce their results with our newly generated LOF alleles. Thanks to the work from Yikang S. Rong’s lab, we could revise our previous conclusions and, overall, we now feel closer to understand dBigH1 function and regulation in embryogenesis. This story is an important reminder of how complex in vivo gene function studies can be, showing the caveats of using technologies as P-element excision to generate mutant conditions. Even when using CRISPR/Cas9 technology, we must be open to recheck our conclusions at any time since this technology does not seem to be as ‘clean’ as anticipated6. The continuous revision of previous models and conclusions is central to our work as scientists.

2) Now that you report this preprint. How are you planning to mitigate this challenge and take it forward?

FA: Currently, we are trying to understand the nature of the strong embryo lethality associated with the bigH1100 allele, something that, we must say, is not being an easy task. In fact, in our initial work3, bigH1100 lethality was significantly rescued by the zygotic expression of an ectopic UAS-dBigH1 construct. According to our current results, it appears that bigH1100 lethality is the consequence of a complex interaction between maternal and zygotic defects. Once these studies are finished, we plan on submitting an extended version of this preprint describing in detail all these observations and discussing the best available tools to study the dBigH1 function. On the other hand, this challenge has opened new and very interesting questions. Unraveling the mechanisms underlying compensatory dH1 expression in dBigH1 LOF backgrounds is one of our main interests.

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