The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actinrelated protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana. We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in droughtresponsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

BackgroundHistone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits.ResultsWe report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.ConclusionsGrowing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0461-1) contains supplementary material, which is available to authorized users.

Nucleosomal acetyltransferase of H4 (NuA4) is an essential transcriptional coactivator in eukaryotes, but remains poorly characterized in plants. Here, we describe Arabidopsis homologs of the NuA4 scaffold proteins Enhancer of Polycomb-Like 1 (AtEPL1) and Esa1-Associated Factor 1 (AtEAF1). Loss of AtEAF1 results in inhibition of growth and chloroplast development. These effects are stronger in the Atepl1 mutant and are further enhanced by loss of Golden2-Like (GLK) transcription factors, suggesting that NuA4 activates nuclear plastid genes alongside GLK. We demonstrate that AtEPL1 is necessary for nucleosomal acetylation of histones H4 and H2A.Z by NuA4 in vitro. These chromatin marks are diminished genome-wide in Atepl1, while another active chromatin mark, H3K9 acetylation (H3K9ac), is locally enhanced. Expression of many chloroplast-related genes depends on NuA4, as they are downregulated with loss of H4ac and H2A.Zac. Finally, we demonstrate that NuA4 promotes H2A.Z deposition and by doing so prevents spurious activation of stress response genes.

B-cell lymphomas and leukemias derive from B cells at various stages of maturation and are the 6th most common cancer-related cause of death. While the role of several oncogenes and tumor suppressors in the pathogenesis of B-cell neoplasms was established, recent research indicated the involvement of non-coding, regulatory sequences. Enhancers are DNA elements controlling gene expression in a cell type- and developmental stage-specific manner. They ensure proper differentiation and maturation of B cells, resulting in production of high affinity antibodies. However, the activity of enhancers can be redirected, setting B cells on the path towards cancer. In this review we discuss different mechanisms through which enhancers are exploited in malignant B cells, from the well-studied translocations juxtaposing oncogenes to immunoglobulin loci, through enhancer dysregulation by sequence variants and mutations, to enhancer hijacking by viruses. We also highlight the potential of therapeutic targeting of enhancers as a direction for future investigation.

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate expression of adjacent genes in a cell type- and developmental stage-specific manner. To date, a comprehensive analysis of direct MYC targets with essential roles in different types of cancer is missing. To enable identification of functional MYC binding sites and corresponding target genes, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. As a proof of principle, using this library we identified several known and novel MYC targets critical for K562 chronic myelogenous leukemia cells and uncovered specific features of essential E-boxes. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in any cancer type.

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NuA4, an essential histone acetyltransferase complex, is required for efficient transcription in eukaryotes. Using genome editing, genomic approaches and biochemical assays, we characterized plant homologues of two key components of this complex, EPL1 and EAF1 in Arabidopsis thaliana. Surprisingly, we found that loss of AtEPL1, which is necessary for enzymatic activity of NuA4, is not lethal. Contrary to yeast, mutants lacking AtEAF1, responsible for complex targeting, display severe pleiotropic phenotype which copies that of Atepl1. Atepl1 and Ateaf1 mutants grow slowly, contain reduced chlorophyll levels and small chloroplasts. We provide evidence that these alterations are not caused by incapacitation of GLK transcription factors, the major regulators of chloroplast development. Using ChIP-seq we show that H4 acetylation levels are dramatically reduced in the chromatin of the Atepl1 mutant, while H3 acetylation remains mostly unchanged. We use our data to define NuA4-dependent genes and show that chloroplast-related genes are significantly overrepresented in this group, consistent with the pale-green phenotypes of the mutants. We propose that NuA4 was adopted in plants to control nuclear-encoded photosynthesis genes.

The transcription factor MYC is a proto‐oncogene with a well‐documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E‐box sequences in the genome to regulate gene expression in a cell‐type‐ and developmental‐stage‐specific manner. To date, a combined analysis of essential MYC‐bound E‐boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E‐box sequences in a genome‐wide fashion. In parallel, we used the Brunello library to knock out protein‐coding genes. We performed high‐throughput screens with these libraries in four MYC‐dependent cancer cell lines—K562, ST486, HepG2, and MCF7—which revealed several essential E‐boxes and genes. Among them, we pinpointed crucial common and cell‐type‐specific MYC‐regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E‐box disruption affects MYC binding, target‐gene expression, and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well‐validated tool opens new possibilities to gain novel insights into MYC‐dependent vulnerabilities in cancer cells.