The BAH domain of BAHD1 is a histone H3K27me3 reader

Histone recognition by reader modules constitutes a major mechanism for epigenetic regulation (Jenuwein and Allis 2001). BAHD1 (bromo adjacent homology domain containing protein 1) is a vertebrate-specific nuclear protein (Fig. S1) involved in gene silencing by promoting hete-rochromatin formation. BAHD1 is characteristic with an N-terminal proline-rich region, a nuclear localization signal motif, and a C-terminal bromo adjacent homology (BAH) domain (Fig. 1A). Previous study revealed that BAHD1 could act as a scaffold protein and tether diverse heterochromatin-associated factors including HP1, MBD1, SETDB1, HDAC5, and several transcriptional factors to trigger facultative heterochromatin formation (Bierne, Tham et al. 2009). Consistent with a " repressive " role, BAHD1 binds to CpG-rich P3 promoter region of IGF2 (insulin-like growth factor II) then represses IGF2 and IGF2 antisense transcription via the recruitment of MBD1 and HDAC5 (Bierne, Tham et al. 2009). Intriguingly, BAHD1 is also involved in host-pathogen interplay. For example, at early L. monocytogenes infection state, BAHD1 forms a complex with TRIM28 and HP1 to repress interferon-stimulated genes, including IFNL1, IFNL2, and IFNL3. At specific infection stages, Listeria secretes a vir-ulence factor, LntA, which could physically interact with BAHD1 to activate interferon (IFN)-stimulated genes (ISGs) (Lebreton, Lakisic et al. 2011). Despite a repressive role of BAHD1, the molecular mechanism underlying BAHD1 heterochromatin targeting remains largely unexplored. BAH domain is an evolutionarily conserved motif which is found in several chromatin-associated proteins such as Sir3, ORC1, Rsc2, ZMET2, and DNMT1. BAH is characteristic of a conserved β-sheet core (typically 9-bladed) flanked with function-specific N-, C-, and β6-β7 insertions. Recent structural and functional studies revealed multi-facet roles of BAH domain in chromatin regulation (Yang and Xu 2013). For example, in yeast, ORC1 BAH could act as a scaffold to mediate ORC1-Sir1 interaction to form a silencing complex; Sir3 BAH could function as a nucleosome-targeting unit to induce heterochromatin formation; moreover, Rsc2 BAH could bind histone H3 and its interaction interface is conserved in a subset of Rsc-like BAH domains (Chambers, Pearl et al. 2013). Interestingly, in metazoan species, ORC1 BAH domain has acquired a histone methylation reader activity and recognizes H4K20me2 to prompt DNA replication licensing (Kuo, Song et al. 2012). BAH domains also exist in DNA methyltransferases of mammalian DNMT1 and plant ZMET2. Noteworthily, ZMET2 but not DNMT1 BAH domain displays histone H3K9me2 binding activity and thus directly mediates a cross-talk between histone and DNA methylations (Du, Johnson et al. 2015). Previous study showed that deletion …


Materials
The full-length gene of human BAHD1 (residues 1-780) was amplified from HEK293 cDNA library by PCR and verified by sequencing. Histone peptides bearing different modifications were synthesized by SciLight Biotechnology, LLC. Anti-H3K27me3 (PTM-622) antibody was obtained from PTM-BioLabs and the anti-GST antibody (M20007) was obtained from Abmart.

Protein expression and purification
The BAH domain encompassing residues 589-780 of human BAHD1 was cloned in to a pSUMOH10 vector (an in house modified vector based on pET28b) containing an N-terminal 10xHis-SUMO tag. All BAH BAHD1 mutants were generated using QuikChange (Stratagene) method and verified by gene sequencing. The recombinant BAHD1 589-780 was overexpressed in E.coli BL21 (DE3). After overnight induction by 0.2 mM isopropyl β-D-thiogalactoside (IPTG) at 16 °C in TB medium, cells were harvested and suspended in buffer: 20 mM Tris, pH 8.5, 0.5 M NaCl, 5% glycerol. After cell lysis and centrifugation, the recombined protein was purified to homogeneity over HisTrap, and the10xHis-SUMO tag was cleaved by ULP1 overnight at 4°C then removed by reloaded onto the HisTrap column. The free BAH BAHD1 protein was collected via size-exclusion chromatography on Superdex G75 column (GE Healthcare) in elution buffer: 20 mM Tris, pH 8.5, 0.5 M (NH) 2 SO 4 , 5% glycerol. The purification procedures for BAH BAHD1 mutants were essentially the same as the wild type protein.

Modified histone peptide array
The modified histone peptide array was preformed essentially as indicated by the manufacturer (Active Motif, Cat 13001&13005). Briefly, the arrays were blocked with 5% skim milk in TTBS buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20 and 1 mM PMSF) overnight and then incubated with 15 μM GST-tagged protein in binding buffer (20 mM Tris, pH 8.5, 300 mM NaCl) for 5 h at 4 °C. The arrays were washed three times for 10 minutes each using TTBS buffer. Added anti-GST antibody (1: 2000 dilution; Cat: CW0085) for 8 at 4 °C, then washed three times for 10 minutes each. Incubated with second antibody (anti-rabbit, 1:4000 dilution) for 1h at RT and then wash three times before ECL reaction.

Fluorescence-based thermal shift assay (TSA)
The TSA was performed with a CFX96 real-time PCR instrument (Bio-Rad). A typical TSA solution was composed of 1 mg/mL BAH BAHD1 protein, Sypro Orange (Invitrogen) and 5 different kinds of salts (500 mM sodium chloride, sodium formate, sodium acetate trihydrate, sodium nitrate, ammonium sulfate) in buffer containing 20 mM Tris, pH 8.5, 5% glycerol.
During TSA assays, all samples were heated from 15°C to 75°C at a rate of 0.5°C per minute.
Protein denaturation was monitored by increased fluorescence signal of Sypro Orange, which captures exposed hydrophobic residues during thermal unfolding. The recorded curves were analyzed by the software CFX-Manager (Bio-Rad). The temperature corresponding to the inflection point was defined as the melting temperature, Tm.

Isothermal titration calorimetry (ITC) measurements
All calorimetric experiments of the wild type or mutant BAH BAHD1 were conducted at 15 °C using a MicroCal iTC200 instrument (GE Healthcare). The BAH BAHD1 samples were dialyzed in the following buffer: 20 mM Tris, pH 8.5, 500 mM (NH) 2 SO 4 and 5% glycerol. Protein concentration was determined absorbance spectroscopy at 280 nm. Peptides were quantified by weighing on a large scale and then aliquoted and freeze-dried for individual use. Acquired calorimetric titration curves were analyzed using Origin 7.0 (OriginLab) using the "One Set of Binding Sites" fitting model. Detailed thermodynamic parameters of each titration were summarized in Supplementary Table S1.

Cell culture and transfection
Human HeLa cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum (Gibco) and supplemented with 100 U/mL penicillin, and 100 μg/mL streptomycin. Plasmid transfection was performed using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Immunofluorescence staining
HeLa cells were fixed in 4% (w/v) formaldehyde in PBS for 10 min, permeabilized in 0.2% Confocal Microscopy (LSM710). Immunofluorescent images were taken as Z-stacks with a DeltaVision image restoration microscope system. Exposure times and settings for deconvolution were constant for all samples to be compared within any given experiment. The image quantification was performed by Imaris (Bitplane).

Deuterium labeling and mass spectra analysis
To prepare for labeling, all protein solutions were brought to a concentration of 180 μM.
For experiments with BAH BAHD1 protein alone, 2 μl of 180 μM protein was diluted with 18 μl of 20 mM Tris, 500 mM (NH) 2 SO 4 , pH 8.5. For experiments with BAH BAHD1 bound to H3 15-42 K27me3 peptide, 2 μl of 180 μM protein was diluted with 18 μl of 20 mM Tris, 500 mM (NH) 2 SO 4 , pH 8.5. To initiate deuterium labeling, 5 μl of each 180 μM protein solution was diluted with 45 μl of labeling buffer (20 mM Tris, 500 mM (NH) 2 SO 4 , 99% D2O, pH 8.5) at room temperature. At 10 minutes, the labeling reaction was quenched by adding 50 μl of ice-cold quench buffer (fomic acid in water solution at pH 1.3, 100% H2O), then immediately put the sample tube on ice. 5μl 1μM pepsin solution was added for digestion. At 5 minutes, the sample was placed into Waters nanoACQUITY UPLC autosampler for injection. 5 5 μl sample was loaded onto a peptide trap (nanoACQUITY UPLC 20-VM trap Symmetry C18 180umx20mm, Waters). A nanoACQUITY UPLC 1.7um BEH 30 C18 75umx100mm column was used to separate digested peptides. A 10% to 40% gradient of acetonitrile over 20 min at a flow rate of 0.35 μl min−1 was used to separate peptides. Both chromatographic mobile phases contained 0.05% (v/v) formic acid. Mass analysis was conducted with a SYNAPT G2-Si mass spectrometer (Waters) equipped with standard ESI source and lockmass correction. Glu-fibrinopeptide was used to maintain the calibration at 3-5 p.p.m. throughout analysis. The mass spectrometer settings were: ESI+ mode; capillary, 3,200 V; cone, 40 V; source temperature, 80 °C; mass acquisition range, 400-1,800 m/z; scan rate, 0.6 scans per s; instrument always collecting data in MSE mode. Peptic peptides were identified using MSE and IdentityE software within ProteinLynx Global Server 3.0.2 (PLGS) (Waters). The deuterium exchange levels were determined by subtracting the centroid mass of undeuterated peptide from the centroid mass of deuterated peptide using HX-Express (Weis, Engen et al. 2006 (Sievers, Wilm et al. 2011) and graphically displayed with ESPript ).The conserved residues are highlighted in red. The secondary structure is drawn based on the structural model of BAHD1. The potential methyl-lysine binding residues are designated by black circle.