Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.

Histone H3 Lys4 methylation (H3K4me) was proposed as a critical component in regulating the gene expression, epigenetic states, and cellular identities1. The biological meaning of H3K4me is interpreted via conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states1,2. The dysregulation of PHD finger has been implicated in a variety of human diseases including cancers and immune or neurological disorders3. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the C-terminal PHD finger of JARID1A or PHF23 (JARID1APHD3, PHF23PHD), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukemias4,5, generated potent oncoproteins that arrested hematopoietic differentiation and induced acute myeloid leukemia (AML). In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukemogenesis. Mutations in PHD fingers that abrogated H3K4me3-binding also abolished leukemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1, Pbx1), and enforced their active gene transcription. Mechanistically, NUP98-PHD fusions act as ‘chromatin boundary factors’, dominating over polycomb-mediated gene silencing to ‘lock’ developmentally crucial loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukemia stem cells. Collectively, our studies represent the first report wherein the deregulation of PHD finger, ‘effector’ of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during development.

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