Protein Cell DOI 10.1007/s13238-016-0243-z Protein & Cell LETTER The BAH domain of BAHD1 is a histone H3K27me3 reader Dear Editor, has acquired a histone methylation reader activity and rec- ognizes H4K20me2 to prompt DNA replication licensing Histone recognition by reader modules constitutes a major (Kuo, Song et al. 2012). BAH domains also exist in DNA mechanism for epigenetic regulation (Jenuwein and Allis methyltransferases of mammalian DNMT1 and plant ZMET2. 2001). BAHD1 (bromo adjacent homology domain contain- Noteworthily, ZMET2 but not DNMT1 BAH domain displays ing protein 1) is a vertebrate-specific nuclear protein histone H3K9me2 binding activity and thus directly mediates a (Fig. S1) involved in gene silencing by promoting hete- cross-talk between histone and DNA methylations Cell rochromatin formation. BAHD1 is characteristic with an (Du, Johnson et al. 2015). Previous study showed that dele- & N-terminal proline-rich region, a nuclear localization signal tion of the C-terminal BAH domain interfered with co-local- motif, and a C-terminal bromo adjacent homology (BAH) ization of BAHD1 with H3K27me3 at nuclear foci in vivo domain (Fig. 1A). Previous study revealed that BAHD1 could (Bierne, Tham et al. 2009), suggesting a role of BAH in act as a scaffold protein and tether diverse heterochromatin- BAHD1 histone H3K27me3 recognition. In order to test this hypothe- associated factors including HP1, MBD1, SETDB1, HDAC5, sis, we recombinantly expressed BAH (aa 589–780) Protein and several transcriptional factors to trigger facultative BAHD1 with an N-terminal GST tag, and carried out modified histone heterochromatin formation (Bierne, Tham et al. 2009). Con- peptide array screening (Fig. 1B). Many black dots of different sistent with a “repressive” role, BAHD1 binds to CpG-rich P3 intensities were detected in the grid, supporting a histone promoter region of IGF2 (insulin-like growth factor II) then binding activity of BAH . In-depth data analysis revealed represses IGF2 and IGF2 antisense transcription via the BAHD1 that all the positive hits can be classified into two major cate- recruitment of MBD1 and HDAC5 (Bierne, Tham et al. 2009). gories: the H3K9me2/3-containing and the H3K27me2/3- Intriguingly, BAHD1 is also involved in host-pathogen inter- containing clusters. Among these hits, H3K27me3-containing play. For example, at early L. monocytogenes infection state, peptides displayed strongest signal, consistent with a pro- BAHD1 forms a complex with TRIM28 and HP1 to repress posed role of BAH in H3K27me3 recognition. interferon-stimulated genes, including IFNL1, IFNL2, and BAHD1 In order to quantitatively characterize the array data, we IFNL3 . At specific infection stages, Listeria secretes a vir- performed isothermal titration calorimetry (ITC) under an opti- ulence factor, LntA, which could physically interact with mized buffer condition that gives a better melting temperature BAHD1 to activate interferon (IFN)-stimulated genes (ISGs) (Tm = 39°C vs. Tm = 35.5°C) of BAH in (Lebreton, Lakisic et al. 2011). Despite a repressive role of optimized unoptimized BAHD1 thermal shift assays (TSA) (Fig. S2). ITC titration revealed a BAHD1, the molecular mechanism underlying BAHD1 dissociation constant (K ) of 15.9 μmol/L between BAH heterochromatin targeting remains largely unexplored. D BAHD1 and H3 K27me3 peptide. By contrast, the binding affinity BAH domain is an evolutionarily conserved motif which is 15-42 dropped to 2.6 mmol/L in the case of H3 K9me3, and no found in several chromatin-associated proteins such as Sir3, 1-15 bindings were observed for H3 R8me2s, H3 K14ac, and ORC1, Rsc2, ZMET2, and DNMT1. BAH is characteristic of a 1-10 1-15 H3 K36me3 peptides (Fig. 1C, left), suggesting K27 site- conserved β-sheet core (typically 9-bladed) flanked with 28-41 specificity. We next explored the methylation-state preference function-specific N-, C-, and β6-β7 insertions. Recent struc- of H3K27 by BAH . Following the loss of methyl tural and functional studies revealed multi-facet roles of BAH BAHD1 groups, the binding affinity dropped 8-fold for H3K27me2 domain in chromatin regulation (Yang and Xu 2013). For (K =116μmol/L), 129-fold for H3K27me1 (K = 2.0 mmol/L) example, in yeast, ORC1 BAH could act as a scaffold to D D and lost completely for unmodified H3K27, suggesting mediate ORC1-Sir1 interaction to form a silencing complex; BAH is a histone H3K27 trimethyllysine-specific reader Sir3 BAH could function as a nucleosome-targeting unit to BAHD1 (Fig. 1C, right). Remarkably, phosphorylation of H3S28 induce heterochromatin formation; moreover, Rsc2 BAH (H3S28ph) dramatically reduced H3K27me3 binding by could bind histone H3 and its interaction interface is conserved 42-fold (K = 680 μmol/L), suggesting a “methyl-phos” binary in a subset of Rsc-like BAH domains (Chambers, Pearl et al. D switch mechanism of BAH (Fischle, Wang et al. 2003). 2013). Interestingly, in metazoan species, ORC1 BAH domain BAHD1 © The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn LETTER Dan Zhao et al. A 1 NLS 589 780 R8me2sK9me3 R2me2sK4acR8me2sK9me3 R2me2aK4acR8me2sK9me3 Pro-rich BAH i-1. i-2. B i-1. K9me2 K9me3K14ac R2me2sK4acR8me2a R2me2aK4acR8me2aK9me2 i-2. R2me2sK4me2R8me2aK9me3 R2me2sK4acR8me2aK9me3 iii. ii. K27me3 R26me2sK27me3 R26me2aK27me3 R26citrK27me3 K27me2 R26me2sK27me2 R26me2aK27me2 R26citrK27me2 C 0.0 Cell - 2.0 & -4.0 BAHD1 BAHD1 K K D (μmol/L) D (μmol/L) -6.0 H3 K27me3 15.9 15–42 H315–42K27me0 N.D. -8.0 H31–15K9me3 2591 H315–42K27me1 2045 Protein Injection (kcal/mol) H31–10R8me2s N.D. H315–42K27me2 116.3 10.0 - H31–15K14ac N.D. H315–42K27me3 15.9 H328–41K36me3 N.D. H315–42K27me3S28ph 680.3 -12.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Molar ratio Molar ratio D E 1915 GFP-BAHD1 H3K27me3 Merge 19 21 27 32 34 42 K H3 ARTKQTARKS--APRKQ--ATKAARKS--TGG--YR Length D 15–42 15.9 19–34 61.3 21–32 123.8 GFP-BAHD1 H3K9me3 Merge F 0.0 - 2.0 - 4.0 BAHD1 K - 6.0 D (μmol/L) - 8.0 H315–42K27me3 15.9 Injection (kcal/mol) H3 K27me3 61.3 - 10.0 19–34 H321–32K27me3 123.8 - 12.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Molar ratio © The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn H3K27me3 readout by BAHD1 LETTER b Figure 1. The BAHD1 BAH is an H3K27me3-binding domain. titration using H315-42K27me3 peptide. As expected, alanine (A) Domain architecture of human BAHD1 protein. (B) A mutation of the aromatic residues disrupted binding between fi modi ed histone peptide array screen probed with GST-tagged BAHBAHD1 and H3K27me3 peptide (Fig. 2C), supporting a BAHBAHD1 domain. Spots were detected by anti-GST antibody. critical role of the aromatic cage in methyllysine recognition. Positive hits were labeled with red boxes and the corresponding The importance of these aromatic residues was further peptides information was annotated on the right. i-1 & i-2, confirmed by immunofluorescence analysis in HeLa cells. As peptides that contain K9me2/me3; ii, peptides that contain shown in Fig. 2D and quantification in Fig. 2E, Y645A, K27me2/me3. (C) Isothermal titration calorimetry (ITC) fitting W667A, and Y669A mutant EGFP-BAHD1 failed to co-lo- curves of BAHBAHD1 domain with indicated histone peptides. N. calize with H3K27me3 compared with the wild type protein, D., not detected. Peptide sequences and complete thermody- thus supporting the functional importance of the aromatic fl namic parameters were listed in Table S1. (D) Immuno uores- cage for heterochromatin targeting by BAHD1 in vivo. cence of EGFP-BAHD1 (full length) transfected HeLa cells In sum, combining peptide array screen, quantitative labeled with H3K27me3 and H3K9me3 antibodies. (E) ITC binding, hydrogen exchange MS, and cellular co-localization fi tting curves of BAHBAHD1 domain titrated with different frames studies, we established that BAHD1 BAH domain is an of the H3K27me3 peptides. (F) Diagram and sequence infor- H3K27me3-specific reader that discriminates against mation of histone H3K27me3 peptides used in the ITC titration. H3K9me3. H3K27me3 often marks facultative heterochro- matin with important functional implications in gene regula- tion, cell differentiation and development (Gaydos, Wang Both H3K9me3 and H3K27me3 are hallmarks for hete- et al. 2014). By contrast, histone H3K9me3 represents a rochromatin and gene silencing (Kim and Kim 2012). To hallmark for constitutive heterochromatin and can be rec- Cell evaluate the in vivo functional distinction between the two ognized by effector proteins such as HP1 to maintain & marks in BAHD1 recruitment, we next performed co-local- structurally condensed chromatin conformation. Our quanti- ization analysis of BAHD1 with H3K9me3 or H3K27me3 in tative ITC assays revealed that the binding affinity of fl HeLa cells by immuno uorescence. As shown in Fig. 1D, BAHBAHD1 to H3K27me3 (KD = 15.9 μmol/L) is more ectopically expressed EGFP-BAHD1 overlapped nicely with than two orders of magnitude stronger than H3K9me3 the punctate staining pattern of H3K27me3 as evidenced by (KD = 2.6 mmol/L). This recognition preference suggests that Protein the yellow appearance of merged signals. By contrast, little distal sequences flanking the K9/K27 consensus “ARKS” overlapping between EGFP-BAHD1 and H3K9me3 was motif contribute to H3K27me3 recognition. In support, an fi observed. This result con rms the functional connection of optimal binding between H3K27me3 and BAHBAHD1 is BAHD1 with H3K27me3 but not H3K9me3 at cellular level. achieved in a long frame of 15–42 but not in shorter segments H3K9me3 and H3K27me3 share a common “ARKS” of 19–34 or 21–32. Exact molecular basis underlying H3 distal sequence motif (Fig.