Specificity of the chromodomain Y chromosome family of chromodomains for lysine-methylated ARK(S/T) motifs

Abstract

Previous studies have shown two homologous chromodomain modules in the HP1 and Polycomb proteins exhibit discriminatory binding to related methyllysine residues (embedded in ARKS motifs) of the histone H3 tail. Methylated ARK(S/T) motifs have recently been identified in other chromatin factors (e.g. linker histone H1.4 and lysine methyltransferase G9a). These are thought to function as peripheral docking sites for the HP1 chromodomain. In vertebrates, HP1-like chromodomains are also present in the chromodomain Y chromosome (CDY) family of proteins adjacent to a putative catalytic motif. The human genome encodes three CDY family proteins, CDY, CDYL, and CDYL2. These have putative functions ranging from establishment of histone H4 acetylation during spermiogenesis to regulation of transcription co-repressor complexes. To delineate the biochemical functions of the CDY family chromodomains, we analyzed their specificity of methyllysine recognition. We detected substantial differences among these factors. The CDY chromodomain exhibits discriminatory binding to lysine-methylated ARK(S/T) motifs, whereas the CDYL2 chromodomain binds with comparable strength to multiple ARK(S/T) motifs. Interestingly, subtle amino acid changes in the CDYL chromodomain prohibit such binding interactions in vitro and in vivo. However, point mutations can rescue binding. In support of the in vitro binding properties of the chromodomains, the full-length CDY family proteins exhibit substantial variability in chromatin localization. Our studies underscore the significance of subtle sequence differences in a conserved signaling module for diverse epigenetic regulatory pathways.

FIGURE 1.

The CDY protein family and their chromodomains. A, schematic representation of the primary structures of CDYL, CDYL2, and CDY. a.a., amino acids. B, sequence alignment of CDY family chromodomains, which are related to HP1 and Polycomb chromodomains. Three stars above the sequence mark the positions of the aromatic cage residues. Secondary structure elements above the sequence correspond to that deduced for CDYL2 (from PDB code 2dnt). The accession codes are: human CDY, Q9Y6F8; Macaque CDY, AJ31484;, human CDYL, Q9Y232; mouse CDYL, Q9WTK2; human CDYL2, AK096185; Macaque CDYL2, AY271718; mouse CDYL2, AK015452; chicken CDYL2, XP_418964; sea urchin CDYL2, XP 781347. C, backbone superposition of the three-dimensional structure of the human CDYL2 chromodomain (green, from PDB code 2dnt) on the structure of the Drosophila HP1 (from PDB code 1kne) chromodomain (pink) bound to an H3K9me3 peptide (black). D, interaction of recombinant human CDY family chromodomains with an H3K9me3 peptide as measured by fluorescence polarization. Averages from at least three independent measurements are plotted. See Table 1 for dissociation constants.

FIGURE 2.

Interaction with methylated lysine residues is an intrinsic property of CDY family proteins. A, interaction of recombinant full-length human CDY and mouse CDYL2 proteins fused to MBP with an H3K9me3 or the corresponding unmodified peptide as measured by fluorescence polarization. Averages from at least three independent measurements are plotted. B, recombinant MBP fusion proteins used for the measurements in panel A were run on SDS-PAGE gels and stained with Coomassie Blue. Arrows indicate the MBP-CDY and MBP-CDYL2 recombinant proteins. Major degradation products co-purifying with the recombinant proteins are indicated. Molecular weight (MW) markers are shown on the left.

FIGURE 3.

Differential nuclear distribution of CDY family proteins. FLAG-tagged human CDY (A), human CDYL (B), or mouse CDYL2 (C) were transiently expressed in NIH3T3 cells. Immunostaining with anti-FLAG-specific antibodies (green) and anti-H3K9me3 (red)-specific antibodies was analyzed by confocal microscopy. The merged image corresponds to the overlay of the two color channels. Yellow areas indicate colocalization sites for CDY family proteins with H3K9me3 modification. Cells of medium expression level representative for the nuclear distribution of the CDY family proteins are shown (see supplemental Fig. S1 for more images). DNA inside the cell nucleus was stained with DAPI and defines areas of high DNA density that are presumed to be heterochromatic. Scale bar, 10 μm.

FIGURE 4.

Point mutations rescue CDYL H3K9me3 binding. A, close-up view of the aromatic cage and surrounding secondary structure elements of the HP1 chromodomain interacting with H3K9me3 (PDB code 1kne). The side chains of E3 and E38 form hydrogen bonds with the backbone of the H3 tail and are solvent-exposed. Residues are numbered according to the sequence alignment in Fig. 1B. B, fluorescence polarization analysis of wild-type (WT) and mutant human CDYL chromodomains interacting with an H3K9me3 peptide. Averages from at least three independent measurements are plotted.

FIGURE 5.

CDYL mutant chromodomain H3K9me3 interaction is sufficient for localization to pericentromeric heterochromatin. FLAG-tagged human CDYL chromodomain mutants A3E,L4F (A), and P1S,P2Q,A3E,L4F (B) were transiently expressed in NIH3T3 cells. Cells were immunostained and analyzed as described in Fig. 3. Cells of medium expression level representative for three different types (I-III) of nuclear distribution found for both mutants are shown. The scale bar is 10 μm. C, the occurrence of type I, II, or III nuclear distribution in each cell for wild-type CDYL (WT), CDYL A3E,L4F, and CDYL P1S,P2Q,A3E,L4F were statistically analyzed. Frequency plots resulting from the evaluation of at least 40 cells each from different transfection experiments are shown.

FIGURE 6.

Interaction of CDY family chromodomains with methyllysines embedded in ARK(S/T) motifs. A, the ARK(S/T) motifs in chromatin that are potential binding sites of CDY family chromodomains are specified. The various methyltransferases that are known to establish the different methylations are listed above each lysine residue. B, fluorescence polarization binding assays were used to determine the specificities of the CDY, CDYL2, and CDYL P1S,P2Q,A3E,L4F chromodomains to each of these peptides containing a trimethyllysine. Averages from at least three independent measurements are plotted.