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.
Highlights
Are associated with spermatogenic failure and male infertility [1, 4, 5]
CDY family proteins have two conserved domains implicated in histone modification and recognition; that is, a chromodomain followed by an enoyl-coenzyme A hydratase/isomerase (ECH) putative catalytic domain (Fig. 1A)
Distinct in Vivo Distribution of CDY, CDYL, and CDYL2 Proteins—Given the CDYL chromodomain does not bind to the H3K9me3 peptide and the CDY chromodomain binds more avidly than the CDYL2 chromodomain, we investigated the in vivo localization of these proteins in relation to heterochromatin in mammalian systems
Summary
Antibodies—Polyclonal antibodies specific for H3K9me were a gift from Dr Thomas Jenuwein (IMP Vienna). Full-length CDY and CDYL2 were cloned into a modified pMAL vector (New England Biolabs) for expression as MBP-His fusion proteins (details are available upon request). To generate C-terminal epitope-tagged constructs for the transient expression of full-length human CDY and human CDYL, we used PCR amplification with reverse primers containing the sequence encoding for the FLAG and hemagglutinin peptides (for details, see Ref. 47). Fluorescence Polarization Binding Assays—Fusion proteins with the N-terminal His tag were expressed in Escherichia coli strain BL21(DE3) (Novagen) and purified by Ni2ϩ-affinity chromatography (Qiagen) and gel filtration chromatography (Superdex 75 resin, GE Healthcare). Fluorescence polarization binding assays were performed under conditions of 20 mM imidazole, pH 8.0, 25 mM NaCl, 2 mM dithiothreitol and in the presence of 100 nM fluorescein-labeled peptide following a previously described protocol [13]. Pictures were taken on a Leica SP5 confocal microscope or a Zeiss Axiopod II both equipped with 60ϫ lenses
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
