Samantha G. Pattenden

A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

An Orally Bioavailable Chemical Probe of the Lysine Methyltransferases EZH2 and EZH1

EZH2 or EZH1 is the catalytic subunit of the polycomb repressive complex 2 that catalyzes methylation of histone H3 lysine 27 (H3K27). The trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive post-translational modification. Overexpression of EZH2 and hypertrimethylation of H3K27 have been implicated in a number of cancers. Several selective inhibitors of EZH2 have been reported recently. Herein we disclose UNC1999, the first orally bioavailable inhibitor that has high in vitro potency for wild-type and mutant EZH2 as well as EZH1, a closely related H3K27 methyltransferase that shares 96% sequence identity with EZH2 in their respective catalytic domains. UNC1999 was highly selective for EZH2 and EZH1 over a broad range of epigenetic and non-epigenetic targets, competitive with the cofactor SAM and non-competitive with the peptide substrate. This inhibitor potently reduced H3K27me3 levels in cells and selectively killed diffused large B cell lymphoma cell lines harboring the EZH2(Y641N) mutant. Importantly, UNC1999 was orally bioavailable in mice, making this inhibitor a valuable tool for investigating the role of EZH2 and EZH1 in chronic animal studies. We also designed and synthesized UNC2400, a close analogue of UNC1999 with potency >1,000-fold lower than that of UNC1999 as a negative control for cell-based studies. Finally, we created a biotin-tagged UNC1999 (UNC2399), which enriched EZH2 in pull-down studies, and a UNC1999-dye conjugate (UNC2239) for co-localization studies with EZH2 in live cells. Taken together, these compounds represent a set of useful tools for the biomedical community to investigate the role of EZH2 and EZH1 in health and disease.

The Proteasome Regulatory Particle Alters the SAGA Coactivator to Enhance Its Interactions with Transcriptional Activators

Promoter recruitment of the Saccharomyces cerevisiae SAGA histone acetyltransferase complex is required for RNA polymerase II-dependent transcription of several genes. SAGA is targeted to promoters through interactions with sequence-specific DNA binding transcriptional activators and facilitates preinitiation-complex assembly and transcription. Here, we show that the 19S proteasome regulatory particle (19S RP) alters SAGA to stimulate its interaction with transcriptional activators. The ATPase components of the 19S RP are required for stimulation of SAGA/activator interactions and enhance SAGA recruitment to promoters. Proteasomal ATPases genetically interact with SAGA, and their inhibition reduces global histone H3 acetylation levels and SAGA recruitment to target promoters in vivo. These results indicate that the 19S RP modulates SAGA complex using its ATPase components, thereby facilitating subsequent transcription events at promoters.

Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia

Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999s on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.

Diverse functions of WD40 repeat proteins in histone recognition

WD40 repeat proteins have been shown to bind the histone H3 tail at the center of their beta-propeller structure. In contrast, in this issue of Genes & Development, Song and colleagues (pp. 1313-1318) demonstrate that the WD40 repeat protein p55 binds a structured region of H4 through a novel binding pocket on the side of beta-propeller, illustrating a diversity of histone recognition by WD40 repeat proteins.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

Histone H4 lysine 16 acetylation breaks the genome's silence

Acetylation at histone H4 lysine 16 is involved in many cellular processes in organisms as diverse as yeast and humans. A recent biochemical study pinpoints this particular acetylation mark as a switch for changing chromatin from a repressive to a transcriptionally active state.

A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1

We report the design and characterization of UNC3866, a potent antagonist of the methyl-lysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb Repressive Complex 1 to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently with a Kd of ∼100 nM for each, and is 6- to 18-fold selective versus seven other CBX and CDY chromodomains while being highly selective versus >250 other protein targets. X-ray crystallography revealed that UNC3866 closely mimics the interactions of the methylated H3 tail with these chromodomains. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform-dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, a known CBX7 phenotype, while UNC4219, a methylated negative control compound, has negligible effects.

Tumor Endothelial Cells with Distinct Patterns of TGFβ-Driven Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) occurs during development and underlies the pathophysiology of multiple diseases. In tumors, unscheduled EndMT generates cancer-associated myofibroblasts that fuel inflammation and fibrosis, and may contribute to vascular dysfunction that promotes tumor progression. We report that freshly isolated subpopulations of tumor-specific endothelial cells (TEC) from a spontaneous mammary tumor model undergo distinct forms of EndMT in response to TGFβ stimulation. Although some TECs strikingly upregulate α smooth muscle actin (SMA), a principal marker of EndMT and activated myofibroblasts, counterpart normal mammary gland endothelial cells (NEC) showed little change in SMA expression after TGFβ treatment. Compared with NECs, SMA(+) TECs were 40% less motile in wound-healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreen(Cdh5-Cre) reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA(+) TECs, suggesting that not all endothelial cells (EC) respond identically to TGFβ in vivo. Indeed, examination of 84 TGFβ-regulated target genes revealed entirely different genetic signatures in TGFβ-stimulated NEC and TEC cultures. Finally, we found that basic FGF (bFGF) exerts potent inhibitory effects on many TGFβ-regulated genes but operates in tandem with TGFβ to upregulate others. ECs challenged with TGFβ secrete bFGF, which blocks SMA expression in secondary cultures, suggesting a cell-autonomous or lateral-inhibitory mechanism for impeding mesenchymal differentiation. Together, our results suggest that TGFβ-driven EndMT produces a spectrum of EC phenotypes with different functions that could underlie the plasticity and heterogeneity of the tumor vasculature.

A chemical tool for in vitro and in vivo precipitation of lysine methyltransferase G9a.

Here we report the design, synthesis, and biochemical characterization of a new chemical tool, UNC0965. UNC0965 is a biotinylated version of our previously reported G9a chemical probe, UNC0638. Importantly, UNC0965 maintains high in vitro potency and is cell penetrant. The biotinylated tag of UNC0965 enables “chemiprecipitation” of G9a from whole cell lysates. Further, the cell penetrance of UNC0965 allowed us to explore the localization of G9a on chromatin both in vitro and in vivo through chemical inhibitor‐based chromatin immunoprecipitation (chem‐ChIP).