Evolutionary adaptation of the chromodomain of the HP1-protein Rhino allows the integration of chromatin and DNA sequence signals
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eLife assessment
The authors use a powerful combination of phylogenetics, structure prediction, biochemistry, and mutagenesis to provide an understanding of the mechanism that provides target specificity of Drosophila HP1 homolog Rhino vs. HP1, with Rhino specifically binding to piRNA loci. The authors show that a single amino acid substitution in the chromodomain of Rhino allows binding of the zinc finger protein Kipferl, which directs the complex to a subset of heterochromatic regions that other HP1 homologs do not. The evidence supporting the conclusions is compelling, providing an impressive level of mechanistic understanding of how the specificity of the piRNA genome defense system is defined. Also, the study highlights how a single amino acid change can change the functionality of a protein, providing fundamental insight into protein evolution.
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Abstract
Members of the diverse heterochromatin protein 1 (HP1) family of proteins play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin binding patterns, presumably due to their interactions with various specificity factors. Here, we elucidate the molecular basis of the protein-protein interaction between the HP1 protein Rhino, a critical factor of the Drosophila piRNA pathway, and Kipferl, a DNA sequence-specific C 2 H 2 zinc finger protein and Rhino guidance factor. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino’s chromodomain, G31D, that does not affect H3K9me2/3 binding but abolishes the specific interaction between Rhino and Kipferl. Flies carrying the rhino G31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhino G31D flies. Thus, Rhino’s chromodomain serves as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.
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eLife assessment
The authors use a powerful combination of phylogenetics, structure prediction, biochemistry, and mutagenesis to provide an understanding of the mechanism that provides target specificity of Drosophila HP1 homolog Rhino vs. HP1, with Rhino specifically binding to piRNA loci. The authors show that a single amino acid substitution in the chromodomain of Rhino allows binding of the zinc finger protein Kipferl, which directs the complex to a subset of heterochromatic regions that other HP1 homologs do not. The evidence supporting the conclusions is compelling, providing an impressive level of mechanistic understanding of how the specificity of the piRNA genome defense system is defined. Also, the study highlights how a single amino acid change can change the functionality of a protein, providing fundamental insight into …
eLife assessment
The authors use a powerful combination of phylogenetics, structure prediction, biochemistry, and mutagenesis to provide an understanding of the mechanism that provides target specificity of Drosophila HP1 homolog Rhino vs. HP1, with Rhino specifically binding to piRNA loci. The authors show that a single amino acid substitution in the chromodomain of Rhino allows binding of the zinc finger protein Kipferl, which directs the complex to a subset of heterochromatic regions that other HP1 homologs do not. The evidence supporting the conclusions is compelling, providing an impressive level of mechanistic understanding of how the specificity of the piRNA genome defense system is defined. Also, the study highlights how a single amino acid change can change the functionality of a protein, providing fundamental insight into protein evolution.
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Joint Public Review:
This article is a direct follow-up to the paper published last year in eLife by the same group. In the previous article, the authors discovered a zinc finger protein, Kipferl, capable of guiding the HP1 protein Rhino towards certain genomic regions enriched in GRGGN motifs and packaged in heterochromatin marked by H3K9me3. Unlike other HP1 proteins, Rhino recruitment activates the transcription of heterochromatic regions, which are then converted into piRNA source loci. The molecular mechanism by which Kipferl interacts specifically with Rhino (via its chromodomain) and not with other HP1 proteins remained enigmatic.
In this latest article, the authors go a step further by elucidating the molecular mechanisms important for the specific interaction of Rhino and not other HP1 proteins with Kipferl. A phylogenetic …
Joint Public Review:
This article is a direct follow-up to the paper published last year in eLife by the same group. In the previous article, the authors discovered a zinc finger protein, Kipferl, capable of guiding the HP1 protein Rhino towards certain genomic regions enriched in GRGGN motifs and packaged in heterochromatin marked by H3K9me3. Unlike other HP1 proteins, Rhino recruitment activates the transcription of heterochromatic regions, which are then converted into piRNA source loci. The molecular mechanism by which Kipferl interacts specifically with Rhino (via its chromodomain) and not with other HP1 proteins remained enigmatic.
In this latest article, the authors go a step further by elucidating the molecular mechanisms important for the specific interaction of Rhino and not other HP1 proteins with Kipferl. A phylogenetic study carried out between the HP1 proteins of 5 Drosophila species led them to study the importance of an AA Glycine at position 31 located in the Rhino chromodomain, an AA different from the AA (aspartic acid) found at the same position in the other HP1 proteins. The authors then demonstrate, through a series of structure predictions, biochemical, and genetic experiments, that this specific AA in the Rhino-specific chromodomain explains the difference in the chromatin binding pattern between Rhino and the other Drosophila HP1 proteins. Importantly, the G31D conversion of the Rhino protein prevents interaction between Rhino and Kipferl, phenocopying a Kipfer mutant.
Strengths:
The authors' effective use of phylogenetic analyses and protein structure predictions to identify a substitution in the chromodomain that allows Rhino's specific interaction with Kipferl is very elegant. Both genetic and biochemical approaches are applied to rigorously probe the proposed explanation. They used a point mutation in the endogenous locus that replaces the Rhino-specific residue with the aspartic acid residue present in all other HP1 family members. This novel allele largely phenocopies the defects in hatch rate, chromatin organization, and piRNA production associated with kipferl mutants, and does not support Kipferl localization to clusters. The data are of high quality, the presentation is clear and concise, and the conclusions are generally well-supported.
Weaknesses:
The reviewers identified potential ways to further strengthen the manuscript.
The one significant omission is RNAseq on the rhino point mutant, which would allow direct comparison to cluster, transposon, and repeat expression in kipferl mutants.
The manuscript would benefit from adding more evolutionary comparisons. The following or similar analyses would help put the finding into a broader evolutionary perspective: i) Is Kipferl's surface interacting with Rhino also conserved in Kipferl orthologs? In other words, are the Rhino-interacting amino acids of Kipferl under any pressure to be conserved? ii) The remarkable conservation of Rhino's G31 is at odds with the arms race that is proposed to be happening between the fly's piRNA pathway proteins and transposons. Does this mean that Rhino's chromodomain is "untouchable" for such positive selection?
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