Discovery of small molecule antagonists of human Retinoblastoma Binding Protein 4 (RBBP4)

RBBP4 is a nuclear WD40 motif-containing protein widely implicated in various cancers and a putative drug target. It interacts with multiple proteins within diverse complexes such as nucleosome remodeling and deacetylase (NuRD) complex and polycomb repressive complex 2 (PRC2), as well as histone H3 and H4 through two distinct binding sites. B-cell lymphoma/leukemia 11A (BCL11A), friend of GATA-1 (FOG-1), plant homeodomain finger protein 6 (PHF6) and histone H3 bind to the top of the donut-shaped seven-bladed β-propeller fold of RBBP4, while suppressor of zeste 12 (SUZ12), metastasis associated protein 1 (MTA1) and histone H4 bind to a pocket on the side of the WD40 repeats of this protein. Here, we report the discovery of the first small molecule antagonists of the RBBP4 top pocket, competing with interacting peptides from proteins such as BCL11A and histone H3. We also determined the first crystal structure of RBBP4 in complex with a small molecule (OICR17251), paving the path for structure-guided design and optimization towards more potent antagonists.

Downregulation of the GHRH/GH/IGF1 axis in a mouse model of Börjeson-Forssman-Lehman syndrome

ABSTRACTBörjeson-Forssman-Lehmann syndrome (BFLS) is an intellectual disability and endocrine disorder caused by plant homeodomain finger 6 (PHF6) mutations. Individuals with BFLS present with short stature. We report a mouse model of BFLS, in which deletion of Phf6 causes a proportional reduction in body size compared with control mice. Growth hormone (GH) levels were reduced in the absence of PHF6. Phf6−/Y animals displayed a reduction in the expression of the genes encoding GH-releasing hormone (GHRH) in the brain, GH in the pituitary gland and insulin-like growth factor 1 (IGF1) in the liver. Phf6 deletion specifically in the nervous system caused a proportional growth defect, indicating a neuroendocrine contribution to the phenotype. Loss of suppressor of cytokine signaling 2 (SOCS2), a negative regulator of growth hormone signaling partially rescued body size, supporting a reversible deficiency in GH signaling. These results demonstrate that PHF6 regulates the GHRH/GH/IGF1 axis.

The chromatin-binding protein PHF6 functions as an E3 ubiquitin ligase of H2BK120 via H2BK12Ac recognition for activation of trophectodermal genes

Epigenetic regulation is important for establishing lineage-specific gene expression during early development. Although signaling pathways have been well-studied for regulation of trophectoderm reprogramming, epigenetic regulation of trophectodermal genes with histone modification dynamics have been poorly understood. Here, we identify that plant homeodomain finger protein 6 (PHF6) is a key epigenetic regulator for activation of trophectodermal genes using RNA-sequencing and ChIP assays. PHF6 acts as an E3 ubiquitin ligase for ubiquitination of H2BK120 (H2BK120ub) via its extended plant homeodomain 1 (PHD1), while the extended PHD2 of PHF6 recognizes acetylation of H2BK12 (H2BK12Ac). Intriguingly, the recognition of H2BK12Ac by PHF6 is important for exerting its E3 ubiquitin ligase activity for H2BK120ub. Together, our data provide evidence that PHF6 is crucial for epigenetic regulation of trophectodermal gene expression by linking H2BK12Ac to H2BK120ub modification.

Binding and allosteric transmission of histone H3 Lys-4 trimethylation to the recombinase RAG-1 are separable functions of the RAG-2 plant homeodomain finger

V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.

MYC Regulates PHF8, Which Promotes the Progression of Gastric Cancer by Suppressing miR-22-3p

Plant homeodomain finger protein 8 (PHF8) has been reported to participate in cancer development and metastasis of various types of tumors. However, little is known about the functional mechanism of PHF8 in gastric cancer (GC). This study aimed to explore the PHF8 expression pattern and function, and the role of the MYC/miRNA/PHF8 axis in GC. PHF8 expression was upregulated in GC tissues and cells as measured using quantitative reverse transcription polymerase chain reaction and western blotting. PHF8 knockdown suppressed the proliferation, migration, and invasion of GC cells, as determined using the CCK-8 assay and Transwell assay. MicroRNA-22-3p targeted PHF8, as verified by a dual-luciferase reporter assay. MYC upregulated the protein expression of PHF8 but had no effect on PHF8 mRNA expression. MYC regulates PHF8 by affecting the stability of miR-22-3p. We identified a novel MYC/miR-22-3p/PHF8 regulatory axis in GC. Therefore, PHF8 may provide a new therapeutic target for patients with GC.

MiR-221 Promotes Hepatocellular Carcinoma Cells Migration via Targeting PHF2

MicroRNAs (MiRNAs), which regulate the gene expression leading to translational inhibition or mRNA degradation, are involved in carcinogenesis and tumor progression. Previous studies have demonstrated that miR-221 was one of the most consistent overexpressed miRNAs in several types of cancer. However, the role of miR-221 in human liver cancer progression is not yet fully elucidated. Levels of miR-221 and plant homeodomain finger 2 (PHF2) expressions in human hepatocellular carcinoma (HCC) tissues and cell lines were detected using western blotting and quantitative real-time PCR (qRT-PCR). Cell migration was studied using the transwell assays. A dual-luciferase reporter system was used to validate the target gene of miR-221. The results indicated that miR-221 promoted HCC cell migration. By performing subsequent systematic bioinformatic analyses, we found PHF2 was the target gene of miR-221 and the direct binding relationship was further validated by dual-luciferase reporter assay. In addition, lower expression of PHF2 promoted HCC cell migration and linked to worse overall survival in HCC patients. Finally, the negative correlation between miR-221 and PHF2 expression levels in HCC specimens was further confirmed. Taken together, our findings implied that miR-221 could be a potential candidate for the therapeutics of HCC metastasis.