Abstract
ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.
Highlights
ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF1)-type containing 8) is a putative chromatin reader/effector harboring a PWWP domain, a bromodomain, and a PHD type zinc finger
Similar interaction of the PBP module of ZMYND8 with purified chromatosome from HeLa cells showed its association to histone H3 and H4 (Fig. 1E). These results clearly indicate that ZMYND8 is a core histone H3/H4-interacting protein having a tight association with chromatin
By performing coimmunoprecipitation assays, we found that ZMYND8 interacts with transcription-initiation-competent RNA pol II S5P but not with transcription elongation/termination-competent RNA pol II S2P complex (Fig. 6A, compare lanes 1 and 3)
Summary
Cell Culture and ATRA Treatment—HeLa and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (Gibco, Invitrogen), and SH-SY5Y cells were maintained in DMEM/F-12 (1:1) (Gibco, Invitrogen). The cells were lysed in Lysis Buffer: 20 mM Tris-HCl (pH 8), 150 mM NaCl, 0.05% Nonidet P-40, 1 mM DTT, 2 mM PMSF, 1ϫ protease inhibitor mixture (EDTA-free). Biotinylated histone peptides were incubated with GST-tagged protein in IP Buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Nonidet P-40, 1 mM DTT) at 4 °C, overnight. Proteins were incubated in an equal molar ratio in binding buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Nonidet P-40, 1 mM DTT), pulled down by pre-blocked glutathione-Sepharose beads, and washed in binding buffer, and the protein complex was analyzed by Western blotting. In the case of titration of ZMYND8 (His-PBP) with H4K16 unmodified and H4K16Ac histone peptides, the data analysis was done following the same theory as reported above, and the non-linear curve fitting was done following Equations 1 and 2. A Z score and permutation or Fisher’s Exact Test p value were calculated to assess over-representation of enriched biological categories
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