Jelly H.M.SofersXuanying LiSusan M.Abmayr Jerry L.Workman*d

1Stowers Institute for Medical Research,Kansas City,MO 64111,USA

2Department of Anatomy and Cell Biology,University of Kansas Medical Center,Kansas City,KS 66160,USA

RESEARCH HIGHLIGHT

Reading and Interpreting the Histone Acylation Code

Jelly H.M.Sofers1,a,Xuanying Li1,2,b,Susan M.Abmayr1,2,c, Jerry L.Workman1,*,d

1Stowers Institute for Medical Research,Kansas City,MO 64111,USA

2Department of Anatomy and Cell Biology,University of Kansas Medical Center,Kansas City,KS 66160,USA

Decades of research has explored the epigenetic control of gene expression and the impact of histone post-translational modifcations(PTMs),such as acetylation,on chromatin remodeling.Indeed,the writers,readers,and erasers of lysine acetylation are increasingly well understood.Recent studies have added crotonylation,butyrylation,and propionylation to the types of acylations by which histones are modifed, and identifed the YEATS protein domain as a critical reader of crotonylation.Now,Haitao Li,David Allis,and their colleagues expand the scope of protein domains capable of reading crotonyl-lysine(Kcr)to include double PHD fnger(DPF) domains.Importantly,the mechanism through which these domains recognize Kcr is quite distinct from their recognition by the YEATS domain[1].In this highlight,we discuss recognition of acylated histones by the bromodomain(BRD),the YEATS domain,and PHD fngers.We contrast the structural basis for their recognition of histones modifed by acetylation and more recently discovered histone crotonylation[2-6].

Roles of histone acylation

Histone acylations are a group of chemical modifcations that occur at lysine residues.Lysine acetylation,the addition of -CH3CO,represents only one class of possible lysine acylations(KC=OR).Structurally larger acylations include propionylation (three-carbon side chain), butyrylation, and crotonylation(both four-carbon side chains).

Fundamental questions include what drives these different modifcations,and what are their biological consequences. The constellation of histone acylations is strongly linked to metabolism.The majority of substrates for histone acylation are acetyl-CoA-linked metabolic intermediates.The activity of acetyl-CoA synthetases increases upon high sirtuin(histone deacetylase;HDAC)activity,which depends on the cellular NAD/NADH ratio[7,8].Thus,the metabolic state of the cell is a strong determinant of what acylations are present at what residues in what combination at a given time.Hence,there is the possibility that the pattern of histone acylation may simply refect the metabolic state of the cell.However,the other possibility is that many histone-acyl PTMs are actively read,and signal for changes in chromatin structure or function.In this light,various acylations could refect a delicate mechanism that fne-tunes gene expression to metabolic need[9,10].

Although histone acetylation and crotonylation globally co-localize and mark open chromatin in resting somatic cells,recent evidence suggests that specifc acylations serve distinct functions.Some acylation marks are differentially susceptible to histone acetyltransferase(HAT)overexpression.Notably, the Kcr signal is not increased upon overexpression of CBP or p300[11],two proteins with intrinsic HAT activity.Moreover,histone acylations have different resistance to HDAC activity,which contributes to the formation of subsets of gene loci marked favorably for gene expression[11,12].Crotonylation is more resistant to removal,and forms a strong transcription activating mark at testis-specifc genes that escape post-meiotic sex inactivation[11,13].At the level of individual histone lysine residues,most lysine residues can be modifed by several different acylations.However,some sites are mutually exclusive for acetyl-lysine(Kac)or Kcr[14].Thus,a better understanding of how these various acyl marks translate into changes in chromatin function is necessary.

Readers of histone acetylation

The reader functions of histone modifying complexes rely on signature domains that specifcally recognize and bind histone marks[15].To date,three domains have been reported to recognize Kac.

BRDs are found in diverse nuclear proteins,including HATs(such as GCN5 and PCAF),ATP-dependent remodeling complexes(such as the SWI/SNF-associated Baz1b and SMARCA2/4),methyltransferases(MLL and ASH1L),and various mediators of transcriptional activity[16].BRDs form a conserved structural fold of four bundled left-handed helices and two inter-helical loops that constitute an aromatic, hydrophobic pocket[2].Kac binding relies on conserved amino acid residues,and is supported by water-mediated hydrogen bonds.[2].

A limited number of YEATS domain-containing proteins are associated with HAT complexes,chromatin-remodeling complexes,or transcription-regulating complexes[17].For example,the YEATS domain of AF9,a component of the human super elongation complex(SEC),binds to acetylated histone H3 with a strong preference for H3K9ac[4,6,18].By adopting an immunoglobulin fold,the AF9 YEATS domain utilizes a serine-lined aromatic‘sandwiching’cage for the specifc readout of Kac within the RK consensus sequence [4].This structure ensures an intimate encapsulation of Kac, which is achieved by relayed hydrogen binding,CH-π interactions,and hydrophobic contacts with a high degree of shape complementarity[4].

A large number of PHD-fnger containing proteins recognize both modifed and unmodifed histones,along with nonhistone proteins[19].A small subclass of double PHD fngers is highly specifc for histone acetylation marks[19].Notably, recognition of H3K14ac requires alignment of the histone peptide across a shared surface of the DPF,as has been described for human DPF3b,a component of the BAF chromatin remodeling complex,and the DPF of the HAT MOZ/ MORF/MYST3[3,20-23].At this interface,the N-terminal residues(R2-K4)from H3 form an anti-parallel β-sheet that contributes to the β-sheet of PHD2.Notably,the MOZ DPF induces an α-helical conformation in the H3 tail,which brings a stretch of N-terminal residues close together for PTM sampling[22].H3K14ac itself interacts with the hydrophobic pocket of PHD1[3,20-23].

Readers of histone crotonylation

Kcr is not only a fairly large acylation mark,it is also distinct from other acylation marks by the π-electron conjugation of its crotonylamide group.This arrangement gives the PTM an extended ligand conformation that is both rigid and planar in shape.Proper recognition requires non-distortion of this planar orientation[6].BRDs[9],YEATS domains[6],and the recently-reported DPF domains[1]all have Kcr binding capacity,albeit through different mechanisms and with different affnities.

The reader pocket of BRDs forms a shaft that is open along the longitudinal axis but has a closed end,a so-called‘side open”confguration(Figure 1A).Low affnity binding to the increasingly longer acylation chains that are added by propionylation,crotonylation,and butyrylation can be accommodated when the extended hydrocarbon chain curls up at the deepest point of insertion.Alternatively,a larger hydrophobic pocket also allows better recognition and binding of these acylations.The gatekeeper residue(fanking the BC-inter-helical loop)determines the size of this pocket.The larger hydrophobic pocket formed with tyrosine gatekeepers(as found in the BRDs of CECR2 and BRD9)accommodates the fexible 4-carbon butyryllysine(Kbu)group.Nevertheless,this pocket still defects the rigid four-carbon Kcr group from being optimally coplanar,contributing to the low affnity of BRD9 for Kcr[9]. In contrast to the BRDs of CECR2 and BRD9,the second BRD of Taf1,later referred to as Taf1(2),can bind to Kbu and Kcr in a co-planar conformation,since these modifcations displace water molecules from their usual positions within the pocket,consistent with a higher-affnity interaction[9].

The YEATS domain,originally identifed as a novel family of Kac reader molecules,has now been found to have a broader acyl-binding capacity and a preference for Kcr over Kac[6,24].The AF9 YEATS binds to H3K9cr or H3K18cr through an aromatic-sandwiching cage,in which the crotonylamide plane is sandwiched by aromatic residues,π-aromatic stacking,and hydrophobic contacts(Figure 1B)[6].In contrast to the BRD reading pocket,the reader pocket of YEATS is elongated and open-ended,increasing its ability to accommodate a large repertoire of acyl-lysine marks with extended acylation chains[6].

Haitao Li,David Allis,and their colleagues[1]now demonstrate that H3K14ac can be recognized and bound by the DPFs in the MOZ/MORF HAT and in the non-catalytic DPF2 subunit of the BAF ATP-dependent nucleosome remodeling complex.Most importantly,these DPFs use a highlyselective pocket to recognize the recently-identifed histone Kcr mark,and bind in a manner that is different from that of the YEATS domain.Combining information from crystal structures,isothermal titration calorimetry,and functional assays,this elegant work describes the salient features of DPF structure and interaction with Kcr.

Figure 1 Schematic overview of the recognition modes of crotonyl-lysine by the bromodomain,YEATS domain,and double PHD fnger domain

In brief,the tandem PHD fngers of MOZ and DFP2 fold compactly into one unit that surveys histone tails and is able to recognize and bind specifc histone acylations.As noted above for the Kac reading mechanism of a single PHD fnger, the β1 surface of PHD2 within MOZ recognizes histone H3R2 and H3K4 within the induced α-helix of the N-terminal part of the histone tail.The actual interaction with H3K14ac relies on the β2 pocket of PHD1.This work is the frst to demonstrate that PHD1 interacts with a second induced α-helix of H3, which might facilitate interactions with various acylations. The β2 surface of PHD1 forms a closed-end hydrophobic pocket that fts like a glove around the rigid and planar crotonylation side chain(Figure 1C).This reader pocket is highly size selective.This ft,shown for H3K14cr,is brought about by substituting bulky residues in the β2 pocket with the side-chain-free glycine.This substitution is conserved among the PHD1 domains of human MOZ,MORF,DPF1, DPF2,DPF3,and PHF10,suggesting a common structure that can better accommodate large side chains.Nonetheless, even proteins with structurally-different DPFs exhibit different binding affnities for Kcr.Notably,DPF2 has a higher binding affnity than MOZ,and mutation analysis demonstrates that substitution of critical residues can convert the binding affnity of MOZ to that of DPF2 and vice versa.Hydrogen bonds between the crotonylamide group and these critical residues trigger a rotational shift of the K14cr side chain and the rearrangement of three water molecules around the carbonyl group of crotonylamide.Reminiscent of Taf1(2)[9],these changes in the confguration of water molecules create a better ft for Kcr. In addition,the actual number of water molecules caninfuence which acylations are bound.This ability to accommodate different acyl-lysines has been shown for the MOZ DPF,where more water molecules in the β2 reader pocket facilitate binding of larger modifcations.One fnal unique feature of DPFs is their ability to bind large acylations with moderate to high affnity,even in the absence of deep pockets (Figure 1C).

Conclusion

BRDs,YEATS domains,and DPFs read various histone acyllysines with different recognition mechanisms and binding affnities.BRDs adopt a‘side-open’pocket that can bind to histone acetyl-lysine with high affnity,but is generally unable to bind to large,rigid residues like crotonylation due to steric hindrance.The YEATS domain uses an‘end-open’aromatic sandwich pocket to bind to acylated amino acid residues, which favors Kcr over Kac.Lastly,the DPFs of MOZ and DPF2 employ a‘dead-end’hydrophobic pocket for Kcr-specifc recognition.Like YEATS domains,DPFs prefer Kcr over Kac,but the basis for this selectivity is different.Specifcally, DPFs exploit intimate hydrophobic encapsulation and coordinated hydrogen bonding,generating a larger pocket and specifc interactions that favor Kcr.

The cell context-dependent constellation of histone modifcations encodes a wealth of information.We are only just beginning to unravel the specifc reading mechanisms.The fact that different domains are able to bind to the same histone acylation brings into question what determines which domain will bind under what circumstances.Thus,dissecting recognition mechanisms for histone acylations will broaden our understanding of how histone modifcations signal for changes in chromatin accessibility and regulate gene expression.

Competing interests

The authors have declared no competing interests.

Acknowledgments

This work was supported by the National Institute of General Medical Sciences(NIGMS;Grant No.R35GM118068)and the Stowers Institute for Medical Research(SIMR)of the United States.

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Received 26 November 2016;revised 1 December 2016;accepted 1 December 2016 Available online 19 December 2016

Handled by Yu Xue

*Corresponding author.

E-mail:JLW@stowers.org(Workman JL).

aORCID:0000-0002-1051-7375.

bORCID:0000-0001-5587-2453.

cORCID:0000-0002-8540-8819.

dORCID:0000-0001-8163-1952.

Peer review under responsibility of Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China.

http://dx.doi.org/10.1016/j.gpb.2016.12.001

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