Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

  1. Jugal Mohapatra
  2. Kyuto Tashiro
  3. Ryan L Beckner
  4. Jorge Sierra
  5. Jessica A Kilgore
  6. Noelle S Williams
  7. Glen Liszczak

  • Curated by eLife

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    Evaluation Summary:

    Poly-ADP-ribosylation (poly-ADPr) is a major histone modification that plays critical roles in DNA damage. However careful mechanistic dissection of the role of poly-ADPr has been challenging as the modification is found on multiple proteins and there is heterogeneity in terms of poly-ADP-ribosylation chain length and amino acid location of attachment. The PARP1-dependent semi-synthetic strategy developed by the authors allows generation of nucleosomes with mono ADP ribose and defined lengths of poly-ADPr chains at specific histone serine residues. The utility of this method is clearly demonstrated by the authors' findings that ALC1, a chromatin remodeler that recognizes poly-ADPr is stimulated substantially by the presence of poly-ADPr on H2A and H3.

    (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

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here, we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at histone H2B serine 6 or histone H3 serine 10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels ‘activated’ substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

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  1. Version published to 10.7554/elife.71502 on eLife
  2. eLife

    Evaluation Summary:

    Poly-ADP-ribosylation (poly-ADPr) is a major histone modification that plays critical roles in DNA damage. However careful mechanistic dissection of the role of poly-ADPr has been challenging as the modification is found on multiple proteins and there is heterogeneity in terms of poly-ADP-ribosylation chain length and amino acid location of attachment. The PARP1-dependent semi-synthetic strategy developed by the authors allows generation of nucleosomes with mono ADP ribose and defined lengths of poly-ADPr chains at specific histone serine residues. The utility of this method is clearly demonstrated by the authors' findings that ALC1, a chromatin remodeler that recognizes poly-ADPr is stimulated substantially by the presence of poly-ADPr on H2A and H3.

    (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.)

  3. eLife

    Reviewer #1 (Public Review):

    Histone modifications play diverse roles in regulating DNA based transactions. Understanding the biochemical mechanisms underlying such roles has been greatly enabled by the ability to reconstitute chromatin templates with defined modifications. Poly-ADP-ribosylation (poly-ADPr) is a major modification that plays critical roles in DNA damage. However a few features have slowed careful mechanistic dissection of the role of poly-ADP-ribosylation. These features include that (i) the modification is found on multiple proteins such as histones and the PARP enzymes that deposit the modification and (ii) there is heterogeneity in terms of the poly-ADP-ribosylation chain length and the amino-acid location of the modification. Additionally, unlike with other histone modifications such as acetylation, methylation and ubiquitylation, generating homogenously modified poly-ADPr chromatin in vitro has been technically challenging. Here the authors generate nucleosomes with homogenously and site-specifically modified poly-ADPr marks through a series of readily separable steps. Using these modified nucleosomes the authors test the impact of the poly-ADPr modifications on the activity of the chromatin remodeler ALC1. Previous work has shown that ALC1 is activated by binding poly-ADPr chains that have 3+ units. However, it has not been clear which modified substrates are being recognized by ALC1. Using their site-specifically modified reagents the authors show clearly that poly-ADP-ribosylation of H2A (and H3) has a much larger effect on stimulating nucleosome remodeling than auto-ADP-ribosylated PARP.

    Overall, the work in this resource article is carefully carried out with several thoughtful controls and provides a highly enabling new method for the chromatin field. What further raises the impact of this work is the use of this new method to make a significant new mechanistic finding about ALC1regulation. I think the work in this resource article will be of much interest to the chromatin community and those studying DNA damage.

  4. eLife

    Reviewer #2 (Public Review):

    This interesting manuscript from Liszczak and coworkers highlights the strength of a semisynthetic chemical approach to obtain both monomeric and well-defined polymeric ADP-ribosylated histones H2B and H3. The authors have applied these protein reagents toward dissecting the role of ADP-ribose in facilitating nucleosome remodeling by the macrodomain-containing remodeler ALC1 in the presence and absence of the PARP1 polymerase. The authors demonstrated a clear preference of ALC1 for longer chains of ADP-ribose and also that ADP-ribosylated PARP1 can stimulate ALC1 activity on nucleosome substrates. Finally, they demonstrated that ADP-ribosylated nucleosomes are reasonable substrates for remodeling in nuclear lysates, and that ALC1-knockout cells do not efficiently remodel ADP-ribosylated nucleosomes suggesting that it is the major remodeler of ADP-ribosylated mononucleosomes. Thus, the manuscript is an elegant demonstration of the utility of histone semisynthesis in testing biochemical mechanisms, such as chromatin remodeling, modulated by histone post-translational modifications. The minor weakness of the current method is that all experiments were done with mononucleosomes alone, which likely do not reflect the distribution of ADP-ribosylation in chromatin. However, some key observations, such as the fact that ADP-ribsoylated PARP1 supports nucleosome remodeling by ALC1 and that ALC1 preferentially remodels mononucleosomes modified with longer ADP-ribose chains are noteworthy and expand our understanding of the roles for histone ADP ribosylation in critical processes such as DNA damage in cells.

  5. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors have developed a PARP1-dependent semi-synthetic strategy to generate mono and poly ADP ribosylated nucleosomes at specific histone serine residues. The authors find thatPARP1/2 interacting protein HPF1 restricts PARP1 activity to favor mono ADP ribosylation and that PARP1 can generate poly ADP ribosylated proteins after HPF1 is removed from 'primed' mono ADP ribosylated substrates. This chemoenzymatic strategy, a close derivative of previously published approach for enzymatic ADP ribosylation, enables generation of hetereogenous PAR chains on peptide substrates, which can be purified to homogeneity by chromatography. The modified peptide is then ligated, using canonical native protein ligation strategies, to the protein of interest. As such, the methodology is well suited for N-terminal modifications of proteins lacking Cys. While this limits broad application of the method, it is well suited for the preparation of modified histones and nucleosomes used in this study.

    Using ADP ribose (ADPr) modified histones, the authors have reconstituted a numbed of nucleosomes with specific lengths and defined sites of ADP ribosylation. These substrates were used to investigate how the chromatin remodeler ALC1 responds to defined lengths and positions of ADP ribosylation, either on nucleosomes or on PARP1 itself. The key finding of the study is that the remodeling activity of ALC1 is dramatically enhanced by poly ADP ribosylation (poly = 4,5), but not mono ADP ribosylation on its nucleosome substrate. This enhancement is greater than that provided by auto ribosylated PARP1 binding to ALC1, which has been previously shown to relieve autoinhibition in ALC1. Furthermore, the stimulatory effect is specific to ALC1 and not to other remodelers tested. Authors further show that the stimulatory effect observed with purified components is also present, albeit to an attenuated extent, in nuclear extracts. Lack of remodeling of modified nucleosomes by extracts of ALC1 knockout cells further implicates this enzyme in remodeling of ADPr modified nucleosomes (of note, this point requires additional analysis - see additional suggestions section).

    While the authors show engagement of the ALC1 macrodomain with polyADP ribosylated histone peptides, the link between macrodomain engagement and enhanced remodeling by the presence of polyADP ribose on the nucleosome substrate is not clearly established. Use of macrodomain mutants that abrogate binding of ADPr, or remodeler deletion constructs would allow for better assessment of whether macrodomain-ADPr engagement is necessary and sufficient for the observed stimulation of the remodeling activity. This would help exclude alternative models by which ADPr of histones can promote remodeling (eg, by potentially impacting chromatin structure or DNA accessibility that might be uniquely suited for ALC1 rather than other remodelers), and might shed light on how ADPr of H3 and H2B yield similar effects, despite different topological constraints.

    Finally, manuscript would benefit from additional contextualization of these findings within the existing knowledge in the field of DNA damage response (DDR). Better understanding of mechanisms that control mono vs polyADP ribosylation (and control chain length for polymerization), nature of HPF1-PARP complex and whether it is constitutive, as well as any prior knowledge that may link HPF1 and ALC1 activity, would help readers further evaluate significance of these findings.

  6. Version published to 10.1101/2021.07.06.449314v1 on bioRxiv