Abstract
The NuRD complex is a conserved transcriptional coregulator that contains both chromatin-remodeling and histone deacetylase activities. Mutations of PHF6 are found in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, or acute myeloid leukemia. Recently, PHF6 was identified to interact with the NuRD complex, and this interaction is mediated by the RBBP4 component. However, little is known about the molecular basis for the interaction. Here, we present the crystal structure of the complex of the NuRD subunit RBBP4 bound to the PHF6 peptide (residues 162-170). The PHF6 peptide binds to the top surface of the RBBP4 β-propeller. A pair of positively charged residues of the PHF6 peptide insert into the negatively charged pocket of RBBP4, which is critical for the interaction between PHF6 and RBBP4. Corresponding PHF6 mutants impair this interaction in vitro and in vivo. Structural comparison shows that the PHF6-binding pocket overlaps with FOG1 and histone H3 on RBBP4/Nurf55, but it is distinct from the pocket recognizing histone H4, Su(z)12, and MTA1. We further show that the middle disordered region (residues 145-207, containing the RBBP4-binding motif) is sufficient for the transcriptional repression mediated by PHF6 on the GAL4 reporter, and knockdown of RBBP4 diminished the PHF6-mediated repression. Our RBBP4-PHF6 complex structure provides insights into the molecular basis of PHF6-NuRD complex interaction and implicates a role for PHF6 in chromatin structure modulation and gene regulation.
Highlights
The PHF6 gene is mutated in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia
In our previous work, we showed that the PHF6 protein can directly interact with the NuRD complex component RBBP4 and that the interaction is mediated by the nucleolar localization sequence (NoLS) composed of NLS3 and NLS4 of PHF6 [20]
PHF6 Binds to RBBP4/RbAp48, Forming a Stable Complex— We previously reported that residues 152–171 of PHF6 can directly interact with RBBP4 (RbAp48) in GST pulldown assays in vitro [20]
Summary
The PHF6 gene is mutated in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, and acute myeloid leukemia. Significance: Association with the NuRD complex implicates a role for PHF6 in chromatin structure modulation and gene regulation. De novo aberrations in PHF6 were identified in female patients with Coffin-Siris syndrome (OMIM 135900) and with a phenotype not identical to Börjeson-Forssman-Lehmann syndrome [15, 16] To study the molecular basis of this interaction, we subsequently determined the crystal structure of RBBP4 in complex with a PHF6 peptide (residues 157–171). We demonstrated that the middle disordered region of PHF6 (residues 145–207, containing the RBBP4-binding motif) is sufficient for transcriptional repression and that the repression is moderately dependent on RBBP4. RBBP4 knockdown by shRNA reduced the PHF6-mediated transcriptional repression
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