Many yeast genes are distinguished by their specific requirements for different components of the transcriptional machinery. Here we examine four genes that fall into two classes as defined by their dependence on specific components of the transcriptional machinery. We describe a series of hybrid constructs, each of which bears activator binding sites that are associated with a promoter other than that with which they are usually affiliated. We examine expression of these reporters in strains bearing three modifications of the transcriptional machinery. Our results indicate that, in each of these cases, the promoter (and not the activator) determines which components of the transcriptional machinery are required. These and additional results, including those of others, clarify how disparate activators can work at many different promoters.

Many genes in bacteria and eukaryotes are activated by "regulated recruitment". According to that picture, a transcriptional activator binds cooperatively to DNA with the transcriptional machinery, and the constitutively active polymerase then spontaneously transcribes the gene. An important class of experiments that helped develop this model is called the "activator by-pass" experiment. In one version of such an experiment, the ordinary activator-transcriptional machinery interaction is replaced by a heterologous interaction. For example, fusing any of several DNA binding domains to Gal11, a component of the yeast mediator complex, creates a powerful activator of genes bearing the corresponding DNA binding sites. Here, we describe a simple modification of the yeast transcriptional machinery that extends the success of similar experiments involving other mediator components. The results reinforce parallels between regulation of enzymes involved in transcription and in other cellular processes.

Chromatin structure plays an essential role in eukaryotic gene expression and cell identity. Traditionally, DNA and histone modifications have been the focus of chromatin regulation; however, recent molecular and imaging studies have revealed an intimate connection between RNA epigenetics and chromatin structure. Accumulating evidence suggests that RNA serves as the interplay between chromatin and the transcription and splicing machineries within the cell. Additionally, epigenetic modifications of nascent RNAs fine-tune these interactions to regulate gene expression at the co- and post-transcriptional levels in normal cell development and human diseases. This review will provide an overview of recent advances in the emerging field of RNA epigenetics, specifically the role of RNA modifications and RNA modifying proteins in chromatin remodeling, transcription activation and RNA processing, as well as translational implications in human diseases.

Eukaryotic transcriptional activators work by recruiting to DNA the transcriptional machinery, including protein complexes required for chromatin modification, transcription initiation, and elongation. Which of these complexes must be directly recruited to trigger transcription? We test various "non-classical" transcription activators (comprising a component of the transcriptional machinery fused to a DNA binding domain) for their abilities to activate transcription of a chromosomally integrated reporter in yeast. Among these newly constructed fusion proteins, none efficiently activated transcription when working on its own. However, in several instances transcription was activated by a pair of such fusion proteins tethered to adjacent sites on DNA. In each of these cases, one fusion protein bore a component of the SAGA complex, and the other bore a component of the Mediator complex. Transcription was also activated by certain tripartite fusion proteins comprising a Mediator and a SAGA component fused to a DNA binding domain. The results are consistent with the finding that the classical activator Gal4, working at the GAL1 promoter, activates transcription by (at least in part) independently recruiting SAGA and Mediator.

Normal hematopoiesis is controlled by a well-connected genetic network composed of several transcription factors (TFs) including PU.1 and GATA1. It has been postulated that both transcription factors and epigenetic modifiers work collaboratively to regulate hematopoietic stem cell differentiation and lineage specification as well as leukemogenesis. However, it is unclear about how the interplay between genetic network and epigenetic regulatory modifiers regulates locus-specific chromatin modifications and gene expression in normal hematopoiesis and hematologic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Drugs targeting epigenetic modifiers including DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and histone deacetylases (HDACs) have been shown to be effective in a small portion of patients with MDS/AML, but the mechanisms underlying the efficacy and selectivity of different epigenetic modifying drugs are unknown. In this study, we performed growth-inhibition experiments with several epigenetic modifying drugs in multiple AML cell lines and identified two distinct lineage/differentiation-associated growth-inhibition patterns. Monocytic leukemia cells, but not erythroid leukemia cells, were sensitive to H3K4 HMT inhibitors, whereas both erythroid and monocytic leukemia cells were hypersensitive to DNMT and H3K27 HMT inhibitors. Importantly, co-immunoprecipitation experiments demonstrated lineage-specific interactions between the lineage-determining TFs (PU.1/SPI1 and GATA1) and the DNA/histone modifiers (DNMT1, DNMT3A/3B, TET2 and EZH2). Specifically, SPI1/PU.1 interacts with DNMT1 and EZH2, while GATA1 interacts with TET2 and DNMT3A/3B in MDS-derived erythroid leukaemia. In monocytic leukemia, SPI1/PU.1 interacts with TET2. Epigenetic modifying drugs such as azacytidine and 3-deazaneplanocin efficiently disrupted the interactions between the lineage-determining TFs and the DNA/histone modifiers without changing the expression of these proteins. We developed a new method, crosslink-assisted DNA modification immunoprecipitation assay (CDMIA), to simultaneously measure 5-methylcytosine (5-mC) and hydroxymethylcytosine (5-hmC). CDMIAs revealed significant drug-responsive changes in 5-mC/5-hmC at the promoters of differentiation/lineage-controlling genes such as PU.1/SPI1, but not at the global 5-mC/5-hmC. Sequential-ChIP and chromatin conformation capture (3C) showed that PU.1/SPI1 recruited polymerase II (pol-II) and the DNA/histone modifying complexes to PU.1/SPI1 toform distinct chromatin structures in a lineage-specific manner. We have selected azacytidine-resistant clones and established azacytidine-resistant cell lines from the previously azacytine-sensitive erythroid and monocytic leukemia cells. Strikingly, azacytine at the same concentrations failed to disrupt the interactions between the lineage-determining transcription factors and the DNA/histone modifiers in these drug-resistant leukemia cells. Genome-wide sequencing revealed novel mutations in TET2, TET3, DNMT3L and PU.1/SP1 that were confirmed by Sanger sequencing. These mutations correlated with the altered interactions between PU.1/SPI1 and the DNA/histone modifying complexes and predicted the responses to epigenetic modifying drugs. Examination of clinical specimens from patients with MDS/AML confirmed the presence of distinct lineage/differentiation-specific chromatin structure with a high-level recruitment of DNA/histone modifiers. Our genome-wide epigenetic analysis demonstrates the statistically significant enrichment of the SPI1/PU.1, TP53 and MYB DNA-binding motifs in hyper-H3K27 trimethylated DNA sequences in erythroid-predominant MDS. These results demonstrate the presence of locus-specific, drug-sensitive chromatin structures in MDS/AML subtypes. Our data revealed a novel epigenetic modifying drug action model that involves selective disruption of the disease-specific interactions between the lineage-determining factors and DNA/histone modifiers. Such drug action models may provide new insights into the mechanisms underlying the efficacy and selectivity of epigenetic modifying drugs. Disclosures Larson: Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy.