A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling.

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions.

High-Throughput Screening and Triage Assays Identify Small Molecules Targeting c-MYC in Cancer Cells.

While c-MYC is well established as a proto-oncogene, its structure and function as a transcription factor have made c-MYC a difficult therapeutic target. To identify small-molecule inhibitors targeting c-MYC for anticancer therapy, we designed a high-throughput screening (HTS) strategy utilizing cellular assays. The novel approach for the HTS was based on the detection of cellular c-MYC protein, with active molecules defined as those that specifically decreased c-MYC protein levels in cancer cells.

EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.

In eukaryotes, post-translational modification of histones is critical for regulation of chromatin structure and gene expression. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). EZH2 overexpression is implicated in tumorigenesis and correlates with poor prognosis in several tumour types.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue.

Wnt3-frizzled 1 chimera as a model to study canonical Wnt signaling.

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low-density lipoprotein receptor-related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF-luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling.

The codon 47 polymorphism in p53 is functionally significant.

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent serine 46 by the proline-directed kinase p38 MAPK is known to greatly enhance the ability of p53 to induce apoptosis.

Methods to study p53-repressed promoters.

There is substantial evidence in the literature that, in addition to functioning as an activator of transcription, the p53 tumor suppressor protein can also function as a sequence-specific transcriptional repressor of a separate set of genes. However, elucidation of the mechanism whereby p53 functions as a transcriptional repressor has been obscured by the use of artificial assays to measure this activity; these assays include transient transfection analyses, where both p53 and target promoters are overexpressed. This chapter describes alternative approaches for the definition of sequence elements that mediate transcriptional repression by p53.

The codon 72 polymorphic variants of p53 have markedly different apoptotic potential.

The gene TP53, encoding p53, has a common sequence polymorphism that results in either proline or arginine at amino-acid position 72. This polymorphism occurs in the proline-rich domain of p53, which is necessary for the protein to fully induce apoptosis. We found that in cell lines containing inducible versions of alleles encoding the Pro72 and Arg72 variants, and in cells with endogenous p53, the Arg72 variant induces apoptosis markedly better than does the Pro72 variant.