Molecular basis for histone H3 “K4me3-K9me3/2” methylation pattern readout by Spindlin1

Abstract

Histone recognition by “reader” modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as “K4me3-R8me2a” and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 “K4me3-K9me3/2” bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 “K4me3-K9me3” peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 “K4me3-K9me3/2” bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions. Histone recognition by “reader” modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as “K4me3-R8me2a” and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 “K4me3-K9me3/2” bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 “K4me3-K9me3” peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 “K4me3-K9me3/2” bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions. Histone posttranslational modifications (PTMs), referred to as the histone codes, are essential for gene regulation and chromatin organization. They are often recognized by cognate reader modules to mediate downstream events (1Jenuwein T. Allis C.D. Translating the histone code.Science. 2001; 293 (11498575): 1074-108010.1126/science.1063127Crossref PubMed Scopus (7633) Google Scholar). In the past two decades, more than 500 histone PTMs have been identified, among which were acetylation, crotonylation, lactylation, methylation, phosphorylation, and ubiquitination. In the meantime, a wealth of “reader” modules, such as Bromo, Chromo, Tudor, MBT, PWWP, WD40, PHD finger, DPF, YEATS, CW finger, ZZZ, and BAH domains, have been characterized to recognize histone PTMs in a type- and site-dependent manner (2Musselman C.A. Lalonde M.E. Côte J. Kutateladze T.G. Perceiving the epigenetic landscape through histone readers.Nat. Struct. Mol. 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Similarly, another bivalent histone methylation signature, H3 “K4me3-K9me3/2,” has been reported to maintain the expression of differentiation master regulatory genes at low levels in trophoblast stem cells, extraembryonic endoderm stem cells, and preadipocytes (10Rugg-Gunn P.J. Cox B.J. Ralston A. Rossant J. Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (20479220): 10783-1079010.1073/pnas.0914507107Crossref PubMed Scopus (173) Google Scholar, 12Matsumura Y. Nakaki R. Inagaki T. Yoshida A. Kano Y. Kimura H. Tanaka T. Tsutsumi S. Nakao M. Doi T. Fukami K. Osborne T.F. Kodama T. Aburatani H. Sakai J. H3K4/H3K9me3 bivalent chromatin domains targeted by lineage-specific DNA methylation pauses adipocyte differentiation.Mol. Cell. 2015; 60 (26590716): 584-59610.1016/j.molcel.2015.10.025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In addition, histone H3K9me3/2 not only exists in regions of silenced genes, but also coexists with H3K4me2 in coding regions of rDNA with active transcription (13Yuan X. Feng W. Imhof A. Grummt I. Zhou Y. Activation of RNA polymerase I transcription by Cockayne syndrome group B protein and histone methyltransferase G9a.Mol. Cell. 2007; 27 (17707230): 585-59510.1016/j.molcel.2007.06.021Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Consistently, the histone H3K9 demethylase PHF8 (KDM7B) has both PHD and JmjC domains and regulates rDNA transcription in nucleolus regions. The PHD domain of PHF8 recognizes H3K4me3 and promotes H3K9me2/1 demethylation by the JmjC domain in cis to further activate rDNA genes (14Feng W. Yonezawa M. Ye J. Jenuwein T. Grummt I. PHF8 activates transcription of rRNA genes through H3K4me3 binding and H3K9me1/2 demethylation.Nat. Struct. Mol. Biol. 2010; 17 (20208542): 445-45010.1038/nsmb.1778Crossref PubMed Scopus (175) Google Scholar). Similarly, the H3K9me3 demethylase JMJD2A (KDM4A) contains a double Tudor domain that recognizes H3K4me3 to balance the genomic distribution of the two marks. On the one hand, the above “read-and-erase” crosstalk supports the co-existence of H3K4me3 and H3K9me3/2 under certain circumstances and, on the other hand, explains the low co-occurrence of H3K4me3 and H3K9me3/2 across our genome (15Huang Y. Fang J. Bedford M.T. Zhang Y. Xu R.M. Recognition of histone H3 lysine-4 methylation by the double Tudor domain of JMJD2A.Science. 2006; 312 (16601153): 748-75110.1126/science.1125162Crossref PubMed Scopus (365) Google Scholar, 16Simon M.D. Chu F. Racki L.R. de la Cruz C.C. Burlingame A.L. Panning B. Narlikar G.J. Shokat K.M. The site-specific installation of methyl-lysine analogs into recombinant histones.Cell. 2007; 128 (17350582): 1003-101210.1016/j.cell.2006.12.041Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). A recent study also found that H3K4me3 and H3K9me3 have some overlaps with obfuscated regulatory roles in Neurospora crassa (17Zhu Q. Ramakrishnan M. Park J. Belden W.J. Histone H3 lysine 4 methyltransferase is required for facultative heterochromatin at specific loci.BMC Genomics. 2019; 20 (31068130): 35010.1186/s12864-019-5729-7Crossref PubMed Scopus (6) Google Scholar). Spindlin1 is a member of Spin/Ssty family and was initially identified as an abundant maternal factor in mouse oocytes and embryos at early developmental stages (18Oh B. Hwang S.Y. Solter D. Knowles B.B. Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo.Development. 1997; 124 (9053325): 493-503Crossref PubMed Google Scholar). Functional studies revealed that Spindlin1 is not only a histone reader protein that regulates downstream gene transcription (19Wang J.X. Zeng Q. Chen L. Du J.C. Yan X.L. Yuan H.F. Zhai C. Zhou J.N. Jia Y.L. Yue W. Pei X.T. SPINDLIN1 promotes cancer cell proliferation through activation of WNT/TCF-4 signaling.Mol. Cancer Res. 2012; 10 (22258766): 326-33510.1158/1541-7786.MCR-11-0440Crossref PubMed Scopus (54) Google Scholar, 20Wang W. Chen Z. Mao Z. Zhang H. Ding X. Chen S. Zhang X. Xu R. Zhu B. Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes.EMBO Rep. 2011; 12 (21960006): PubMed Scopus Google but also is in during or cell S. J. Li 1 is essential for maintenance and in PubMed Scopus Google Scholar, P. B. Yuan H. Chen L. Y. C. X. Shi S. Yue W. Pei X. of and Cell. PubMed Scopus Google Scholar, M. Li Z. distribution of in and from oocytes to and its function for transition in A 2010; PubMed Scopus Google Scholar). In addition, Spindlin1 is in a number of (19Wang J.X. Zeng Q. Chen L. Du J.C. Yan X.L. Yuan H.F. Zhai C. Zhou J.N. Jia Y.L. Yue W. Pei X.T. SPINDLIN1 promotes cancer cell proliferation through activation of WNT/TCF-4 signaling.Mol. 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Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes.EMBO Rep. 2011; 12 (21960006): PubMed Scopus Google Scholar, Wang W. Wang Y. Wang M. Q. Z. Zhu B. Xu R.M. Distinct of lysine-4 of histone H3 recognition by domains of Natl. Acad. Sci. U. S. A. 2012; PubMed Scopus Google Scholar, X. Zhu X. Li H. W. Nucleolar signal and histone methylation reader function is required for to rRNA gene Res. Commun. 2018; PubMed Scopus (6) Google Scholar). Here we characterized Spindlin1 as a potent reader for bivalent H3 “K4me3-K9me3/2” methylation pattern. studies revealed combinatorial readout of and by Moreover, our and revealed genomic of Spindlin1, and In some Spindlin1 and H3K4me3 as peaks in the H3K9me3 suggesting that Spindlin1 is to chromatin primarily through The that H3K9me3/2 further a of Spindlin1 with H3 calls attention to a of Spindlin1 in regulation of gene expression in H3K9me3/2-enriched regions. Spindlin1 has been characterized as a histone reader for a H3 in processes of and rDNA transcription X. Zhu Ding X. S.Y. Y. Zhu B. W. Li H. histone H3 methylation readout by Spin/Ssty of Dev. 2014; PubMed Scopus Google Scholar, X. Zhu X. Li H. W. Nucleolar signal and histone methylation reader function is required for to rRNA gene Res. Commun. 2018; PubMed Scopus (6) Google Scholar). Here we to the histone H3 “K4me3-K9me3/2” methylation be recognized by Spindlin1 or not we using with histone H3 methylation “K4me3-K9me3” peptide the most with a of which is and than of and These that H3K9me3/2 is a recognition mark for Spindlin1 and its with H3K4me3 can we titration to these with the binding affinity of for peptide the binding affinity is to nm for H3 “K4me3-R8me2a” and 16 nm for H3 “K4me3-K9me3” methylation Moreover, we binding of 16 17 and 20 nm between and H3 and The binding affinity between and “K4me3-K9me3/2” with the of the suggests that Spindlin1 is a potent reader for histone H3 “K4me3-K9me3/2” bivalent methylation the for H3 “K4me3-K9me3/2” recognition by Spindlin1, we the structure of Spindlin1 to “K4me3-K9me3” peptide at to Spindlin1 is of three Spin/Ssty that are in a were to H3 to to the The H3 peptide in the of Spindlin1 with an to In the the two methylation H3K4me3 and H3K9me3, are into the aromatic pockets of the and the Tudor domains of Spindlin1, respectively. The aromatic in Tudor 2 is by and and the aromatic in Tudor 1 is of and A and and in are for of of in are for in a we also the structure of Spindlin1 to peptide at H3 to were in the structure Consistently, the H3K4me3 and marks are into the aromatic of the and the Tudor domains of Spindlin1 a of between the two In the the H3 is on the of Spindlin1 by a of or between Spindlin1 and the H3 These are by and In is by and binding are by an affinity of for and for The two methylation marks H3K4me3 and H3K9me3 are by aromatic pockets with of and In the the is for the aromatic pockets recognize lysine 4 or 9 through and we generated of the aromatic in 2 and of the aromatic in Tudor 2 in an affinity of and for and of most aromatic in pocket 1 binding to that binding Collectively, these that recognition of by pocket 2 recognition by pocket 1 binding and a more from the H3 peptide, the structure of the H3 “K4me3-K9me3” is the same as that of the H3 “K4me3-R8me2a” with a of It is that the same pocket of Spindlin1 is for both and H3K9me3/2 mark readout In the H3 “K4me3-R8me2a” structure reported the of H3 peptide is and the and marks are into reader to of H3K9me3/2 into pocket the H3 peptide on a more with an in the and the H3K9me3/2 mark is into aromatic from the from pocket 1 and is by with and The aromatic of Tudor 1 is to H3K9me3 which in of and with from the the of H3K9me3 a by the aromatic which is with a of for as with an of for the of mark by aromatic pocket 1 of Spindlin1 is also with a and of Spindlin1 with the H3 the genomic distribution of Spindlin1, and H3K9me3, we by in cells with and of the peaks showed a of Spindlin1 and in which Spindlin1 peaks with with H. H. D. L. C.A. M. R. R. The histone reader in 2015; PubMed Scopus Google Scholar). on the H3K4me3 and H3K9me3 are in genomic regions with roles in gene we found of H3K4me3 peaks with these peaks are occupied by Spindlin1 distribution further revealed that of the peaks are in the gene regions characterized genes in the on biological showed that more than of the genes are in gene transcription and its and for zinc finger epigenetic and and on from the genes in the gene of the genes are and our studies showed that H3 “K4me3-K9me3/2” and Spindlin1 also in regions of rDNA with a reported function of Spindlin1 in nucleolus the of H3K9me3 peaks in from to more than H3K9me3 of as a we the peaks into two H3K9me3 and H3K9me3 regions. The H3K9me3 group of peaks by H3K9me3, and genes RNA gene and developmental signal protein gene The of the peaks are H3K9me3 among which the rDNA gene and transcription gene the of H3K9me3 recognition in binding we for Spindlin1 and the H3K9me3 pocket at two genome the with H3K9me3 and the with H3K9me3 shown in and are expressed at a in cells, we found that the of in these two regions was with that of suggesting a of recognition for of Spindlin1 in that Spindlin1 has a with H3K4me3 in genomic distribution and binding affinity for histone H3 “K4me3-K9me3/2” methylation we that Spindlin1 is a downstream effector of H3K4me3 in H3K9me3/2-enriched and the H3 “K4me3-K9me3/2” a of Spindlin1 with chromatin to target gene Previous studies have revealed that H3K4me3 be recognized by a of modules, such as Chromo, Tudor, and domains. Here we H3K4me3 the PHD domains of and the domain of and the double Tudor domains of and JMJD2A and binding H3K4me3 and H3 we binding of these for H3K4me3 and H3 “K4me3-K9me3” with and for and for and for and for and and for JMJD2A respectively. found that the of H3K9me3 is by the H3K4me3 with to affinity for the H3 “K4me3-K9me3” In H3K9me3 further promotes by both to 16 nm and by to that Spindlin1 is a and potent histone reader for bivalent histone H3 “K4me3-K9me3” pattern. Histone PTMs and readout roles in such as and In histone PTMs often in a combinatorial which calls for a mechanism of combinatorial readout by reader modules A.J. Li H. Patel D.J. Allis C.D. of chromatin modifications by binding Mol. Biol. 2007; PubMed Scopus Google Scholar). Here we found that Spindlin1 can as a potent reader for histone H3 “K4me3-K9me3/2” methylation pattern. studies revealed that H3K4me3 and H3K9me3/2 are recognized by the aromatic of 2 and 1 of Spindlin1, respectively. and studies that H3K4me3 serves as the binding for recognition and and H3K9me3/2 a with affinity and B and T. A of binding in a PubMed Scopus Google Scholar). The and of many histone PTMs have been well histone heterochromatin a major of cell and during early C. X. Y. L. Li C. W. Chen C. X. Y. Chen J. Wang Y. R. Wang H. T. Zhang Y. of heterochromatin during Biol. 2018; 20 PubMed Scopus Google Scholar). in most the genomic of H3K4me3 and H3K9me3 are we revealed that a of the peaks of H3K4me3 and H3K9me3 as well as Spindlin1 These regions genes of zinc finger and developmental signal these genes are in biological processes such as transcription and its regulation suggesting the of the H3 “K4me3-K9me3” bivalent mark and its It has been reported that most histone H3K9 and were to H3K4me3 mark and were H3K4me3 peptide was as D.J. Garcia B.A. S. of histone H3 lysine 4 methylation of histone H3 lysine regulation of lysine by with 2010; PubMed Scopus Google Scholar). suggests that H3K4me3 is an mark of H3K9 domain has been shown to be of H3K4 in the of H3K9 methylation A. S. S. B. the core a for histone H3 lysine 4 Biol. Chem. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). H3K4me3 can be in regions to a bivalent H3 “K4me3-K9me3” methylation for Spindlin1 as well as the maintenance of a “poised” chromatin The above is also with the of H3K9me3/2 that H3K4me3 reader such as JMJD2A and to target genes from a mechanism be required to the bivalent H3 “K4me3-K9me3” and to other downstream H3K4me3 as to activate gene In the as a maternal factor and a Spindlin1 function as a reader that in H3K9me3/2-enriched heterochromatic regions in to H3K4me3 as to balance gene expression and silencing in processes of early and a in Spindlin1 or was on a with an were in with at in the cells were and in 20 cell and the was a affinity and with the were to by The Spindlin1 was for further by an by a were to in 20 and at for Spindlin1 were generated by the Spindlin1 were using the same as histone H3 were from and “K4me3-K9me3/2” were in of and at for 1 The of the “K4me3-K9me3/2” was using the were generated by of with of at were under the for and for “K4me3-K9me3” respectively. were and in the and in for further were at at were and using the were by in using the Spindlin1 structure as a and were with P. K. for Biol. 60 PubMed Scopus Google and Chen G.J. A.J. R. A for structure Biol. 2010; PubMed Scopus Google respectively. and are in Calorimetric were at with a The were the 20 was by at were by on a was with 17 of the peptide in titration were and with the using the of The was with a was with 60 of the histone peptide and were in of the same and at for 1 were from to at a of was by signal of which the of the protein core during in the signal were and by the of the protein is the and of the protein are in and the is the cells were with using The was as cells were with for 10 at and the was with 125 chromatin and the was and to at using 4 of H3K4me3 and H3K9me3 respectively. were for 4 with of a of at were and at for the DNA was further and for The for are rDNA rDNA For the were with DNA and on to the of to the to the to we the using with the of for and for H3K4me3 and The downstream of peaks was using The and structure of “K4me3-K9me3” and and have been in The were in the at of the for in posttranslational modification DNA titration gene

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