Features of Cryptic Promoters and Their Varied Reliance on Bromodomain-Containing Factors

The Set2-Rpd3S pathway is important for the control of transcription memory. Mutation of components of this pathway results in cryptic transcription initiation within the coding region of approximately 30% of yeast genes. Specifically, deletion of the Set2 histone methyltransferase or Rco1, a component of the Rpd3S histone deacetylase complex leads to hyperacetylation of certain open reading frames (ORFs). We used this mutant as a system to study the role of histone modifications and co-activator recruitment in preinitiation complex (PIC) formation. Specifically, we looked at the dependence of promoters on the bromodomain-containing RSC complex and the Bdf1 protein. We found that the dependence of cryptic promoters for these proteins varied. Overall, our data indicate that cryptic promoters are independently regulated, and their activation is dependent on factors that govern gene activation at canonical promoters

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

Transcriptional regulators lacking enzymatic activity or binding pockets with targetable molecular features have typically been considered “undruggable,” and a reductionist approach based on identification of their molecular targets has largely failed. We have demonstrated that the Ewing sarcoma chimeric transcription factor, EWSR1-FLI1, maintains accessible chromatin at disease-specific regions. We adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), an assay for accessible chromatin, to screen an epigenetically targeted small molecule library for compounds that reverse the disease-associated signature. This approach can be applied broadly for discovery of chromatin-based developmental therapeutics and offers significant advantages because it does not require the selection of a single molecular target. Using this approach, we identified a specific class of compounds with therapeutic potential

A chemical tool for chemiprecipitation of the lysine methyltransferase, G9a, in vitro and in vivo

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

A Role for Widely Interspaced Zinc Finger (WIZ) in Retention of the G9a Methyltransferase on Chromatin

G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation

Tumor Endothelial Cells with Distinct Patterns of TGF -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. Whereas some TEC strikingly up-regulate alpha 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 NEC, SMA+ TEC were 40 % less motile in wound healing assays and formed more stable vascular-like networks in vitro when challenged with TGFβ. Lineage tracing using ZsGreenCdh5-Cre reporter mice confirmed that only a fraction of vessels in breast tumors contain SMA+ TEC, 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 up-regulate others. EC 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

MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing

Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts

Infrequently transcribed long genes depend on the Set2/Rpd3S pathway for accurate transcription

The presence of Set2-mediated methylation of H3K36 (K36me) correlates with transcription frequency throughout the yeast genome. K36me targets the Rpd3S complex to deacetylate transcribed regions and suppress cryptic transcription initiation at certain genes. Here, using a genome-wide approach, we report that the Set2–Rpd3S pathway is generally required for controlling acetylation at coding regions. When using acetylation as a functional readout for this pathway, we discovered that longer genes and, surprisingly, genes transcribed at lower frequency exhibit a stronger dependency. Moreover, a systematic screen using high-resolution tiling microarrays allowed us to identify a group of genes that rely on Set2–Rpd3S to suppress spurious transcripts. Interestingly, most of these genes are within the group that depend on the same pathway to maintain a hypoacetylated state at coding regions. These data highlight the importance of using the functional readout of histone codes to define the roles of specific pathways

Chromatin remodeling controls Kaposi's sarcoma-associated herpesvirus reactivation from latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of three human malignancies, the endothelial cell cancer Kaposi's sarcoma, and two B cell cancers, Primary Effusion Lymphoma and multicentric Castleman's disease. KSHV has latent and lytic phases of the viral life cycle, and while both contribute to viral pathogenesis, lytic proteins contribute to KSHV-mediated oncogenesis. Reactivation from latency is driven by the KSHV lytic gene transactivator RTA, and RTA transcription is controlled by epigenetic modifications. To identify host chromatin-modifying proteins that are involved in the latent to lytic transition, we screened a panel of inhibitors that target epigenetic regulatory proteins for their ability to stimulate KSHV reactivation. We found several novel regulators of viral reactivation: an inhibitor of Bmi1, PTC-209, two additional histone deacetylase inhibitors, Romidepsin and Panobinostat, and the bromodomain inhibitor (+)-JQ1. All of these compounds stimulate lytic gene expression, viral genome replication, and release of infectious virions. Treatment with Romidepsin, Panobinostat, and PTC-209 induces histone modifications at the RTA promoter, and results in nucleosome depletion at this locus. Finally, silencing Bmi1 induces KSHV reactivation, indicating that Bmi1, a member of the Polycomb repressive complex 1, is critical for maintaining KSHV latency