An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability
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eLife assessment
This study shows that cis H3 tail acetylation promotes nucleosome accessibility to H3K4 methyl readers and writers such as MLL1. The findings provide a molecular basis for the long-standing connection between H3 acetylation and H3K4 methylation. Additional evidence is required to fully support the conclusions.
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Abstract
In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation ‘chromatin switch’ on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.
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Author Response
Reviewer #2 (Public Review):
- It has been reported that PHD fingers can bind to DNA in addition to lysine-methylated histone H3. Can the authors address whether or not the enhanced selectivity of PHD-nucleosome interactions over PHD-peptide interactions is due to PHD-DNA binding?
We apologize for not making this clearer in our initial manuscript. We did test the ability of our PHD readers to bind nucleosomal DNA of various lengths and observed no significant engagement (Figure 1 - figure supplement 1B). This is emphasized in the revised text.
- What's the binding affinities of PHD-nucleosome interactions and PHD-peptide interactions, respectively?
The relative EC50 (EC50rel) for these interactions (Figure 1 - figure supplement 1C-D and Figure 2 - figure supplement 1H) are consistent with others using Alpha/dCypher …
Author Response
Reviewer #2 (Public Review):
- It has been reported that PHD fingers can bind to DNA in addition to lysine-methylated histone H3. Can the authors address whether or not the enhanced selectivity of PHD-nucleosome interactions over PHD-peptide interactions is due to PHD-DNA binding?
We apologize for not making this clearer in our initial manuscript. We did test the ability of our PHD readers to bind nucleosomal DNA of various lengths and observed no significant engagement (Figure 1 - figure supplement 1B). This is emphasized in the revised text.
- What's the binding affinities of PHD-nucleosome interactions and PHD-peptide interactions, respectively?
The relative EC50 (EC50rel) for these interactions (Figure 1 - figure supplement 1C-D and Figure 2 - figure supplement 1H) are consistent with others using Alpha/dCypher technologies (e.g. doi.org/10.1101/ 2022.02.21.481373v1; which also contains a detailed description of EC50rel calculation and the difference between this value and an equilibrium Kd).
- Histone H4K5acK8ac is a well-known site-specific histone acetylation mark for gene transcriptional activation, much more so than histone H3 acetylation. Does H4K5K8 acetylation enhance PHD-H4K3me3 binding in nucleosome?
We appreciate the reviewer for asking this question. In our studies, we tested H4K5ac and H4K8ac binding individually but do not have a nucleosome with the dual H4K5ac8ac nucleosome. Given the limited amount of time and resources we had for making more nucleosomes, we felt our efforts were better spent on developing heterotypic nucleosomes to answer the more striking cis vs. trans question posed by both reviewers.
- The authors provided the data showing cis histone H3 tail lysine acetylation effects on PHDH4K3me3 binding. What about trans histone H3 lysine acetylation effects?
Thank you for this suggestion. To address this, we expended considerable resources to create new fully PTM-defined heterotypic (to accompany our homotypic) nucleosomes (note nomenclature to minimize confusion with asymmetric/symmetric DNA methylation) to directly test whether MLL1’s activity enhancement in the context of H3 tail acetylation occurs in cis or in trans. As shown in Figure 2D, enhancement of H3K4 methylation only occurs with heterotypic nucleosomes that have an available H3K4 residue with tail acetylation in cis (H3K4me3 • H3K9acK14acK18ac (hereafter H3triac)) and is not seen in H3K4 methylatable nucleosomes with tail acetylation in trans (H3 -> H3K4acK9acK14acK18ac (hereafter H3tetraac)). These exciting new findings greatly strengthen our study and provide more definitive mechanistic details of H3ac → H3K4me regulation.
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eLife assessment
This study shows that cis H3 tail acetylation promotes nucleosome accessibility to H3K4 methyl readers and writers such as MLL1. The findings provide a molecular basis for the long-standing connection between H3 acetylation and H3K4 methylation. Additional evidence is required to fully support the conclusions.
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Reviewer #1 (Public Review):
Histone peptides have been the primary tools for identification and characterization of histone readers. However, in vivo the real substates of histone readers are nucleosomes, in which histone tails exist in dynamic equilibrium between free, accessible state and DNA-bound, inaccessible states. Therefore, other histone modifications, particularly acetylation, impact the accessibility of histone tails to reader proteins. Using modified nucleosomes and known H3K4me3-binding PHD fingers, the authors show that indeed acetylation on nucleosomes has more profound impact than on histone peptide in terms of binding affinity and specificity, likely through increasing H3K4me3 accessibility. The authors further extend the study to investigate the impact of nucleosomal acetylation on H3K4 methyltransferase MLL1C's …
Reviewer #1 (Public Review):
Histone peptides have been the primary tools for identification and characterization of histone readers. However, in vivo the real substates of histone readers are nucleosomes, in which histone tails exist in dynamic equilibrium between free, accessible state and DNA-bound, inaccessible states. Therefore, other histone modifications, particularly acetylation, impact the accessibility of histone tails to reader proteins. Using modified nucleosomes and known H3K4me3-binding PHD fingers, the authors show that indeed acetylation on nucleosomes has more profound impact than on histone peptide in terms of binding affinity and specificity, likely through increasing H3K4me3 accessibility. The authors further extend the study to investigate the impact of nucleosomal acetylation on H3K4 methyltransferase MLL1C's activity on nucleosome. Surprisingly, MLL1C shows no or very low level of enzymatic activity toward the unacetylated nucleosome, whereas H3 tr-acetylation strongly enhances H3K4 methylation by MLL1C, likely through increasing the accessibility of H3 tail to the enzyme. Consistent with this in vitro data, MS analysis of MCF-7 cells shows that increasing histone acetylation by HDAC inhibitor increases the global levels of H3K4 methylation, particularly on the histone tails with higher levels of acetylation. Together, these findings suggest a model in which acetylation releases nucleosome-bound H3 tails to available H3K4 methyltransferases for subsequent methylation and provide a molecular basis for the long-standing connection between H3 acetylation and H3K4 methylation.
The effect of histone acetylation on histone tail accessibility to reader proteins have been previously reported by several groups, including studies from some of the authors of the current manuscript. The novelty of this study lies on the findings that tail accessibility also affects the enzymatic activity of H3K4 methyltransferases. However, additional evidence is needed to further strengthen the findings.
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Reviewer #2 (Public Review):
In this manuscript, Jian et al. reported their biochemical study demonstrating that histone H3 lysine acetylation facilitates H3K4me3 binding by the PHD finger proteins on nucleosome as compared to peptides, and enhances H3K4 methylation by histone methyltransferase MLL1. Histone lysine acetylation and methylation are well known to play an important role in directing gene transcription in chromatin, but how they work in coordination is much less understood. Therefore, this study provides new insights into how histone H3 lysine acetylation promotes gene transcriptional activation through enhancing writing and reading of histone H3K4 methylation, which is recognized as a histone mark for transcriptional activation. While this is an interesting study, there are a number of questions that the authors should …
Reviewer #2 (Public Review):
In this manuscript, Jian et al. reported their biochemical study demonstrating that histone H3 lysine acetylation facilitates H3K4me3 binding by the PHD finger proteins on nucleosome as compared to peptides, and enhances H3K4 methylation by histone methyltransferase MLL1. Histone lysine acetylation and methylation are well known to play an important role in directing gene transcription in chromatin, but how they work in coordination is much less understood. Therefore, this study provides new insights into how histone H3 lysine acetylation promotes gene transcriptional activation through enhancing writing and reading of histone H3K4 methylation, which is recognized as a histone mark for transcriptional activation. While this is an interesting study, there are a number of questions that the authors should address as described below, which would confirm the functional importance and relevance of their results.
Specific Comments:
1. It has been reported that PHD fingers can bind to DNA in addition to lysine-methylated histone H3. Can the authors address whether or not the enhanced selectivity of PHD-nucleosome interactions over PHD-peptide interactions is due to PHD-DNA binding?
2. What's the binding affinities of PHD-nucleosome interactions and PHD-peptide interactions, respectively?
3. Histone H4K5acK8ac is a well-known site-specific histone acetylation mark for gene transcriptional activation, much more so than histone H3 acetylation. Does H4K5K8 acetylation enhance PHD-H4K3me3 binding in nucleosome?
4. The authors provided the data showing cis histone H3 tail lysine acetylation effects on PHD-H4K3me3 binding. What about trans histone H3 lysine acetylation effects?
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