Essential Functions of the Histone Demethylase Lid

Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C5HC2 zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development

The PRC2-associated factor C17orf96 is a novel CpG island regulator in mouse ES cells

CpG islands (CGIs) are key DNA regulatory elements in the vertebrate genome and are often found at gene promoters. In mammalian embryonic stem (ES) cells, CGIs are decorated by either the active or repressive histone marks, H3K4me3 and H3K27me3, respectively, or by both modifications (‘bivalent domains’), but their precise regulation is incompletely understood. Remarkably, we find that the polycomb repressive complex 2 (PRC2)-associated protein C17orf96 (a.k.a. esPRC2p48 and E130012A19Rik) is present at most CGIs in mouse ES cells. At PRC2-rich CGIs, loss of C17orf96 results in an increased chromatin binding of Suz12 and elevated H3K27me3 levels concomitant with gene repression. In contrast, at PRC2-poor CGIs, located at actively transcribed genes, C17orf96 colocalizes with RNA polymerase II and its depletion leads to a focusing of H3K4me3 in the core of CGIs. Our findings thus identify C17orf96 as a novel context-dependent CGI regulator

The oxidative demethylase ALKBH3 marks hyperactive gene promoters in human cancer cells

Background: The oxidative DNA demethylase ALKBH3 targets single-stranded DNA (ssDNA) in order to perform DNA alkylation damage repair. ALKBH3 becomes upregulated during tumorigenesis and is necessary for proliferation. However, the underlying molecular mechanism remains to be understood. Methods: To further elucidate the function of ALKBH3 in cancer, we performed ChIP-seq to investigate the genomic binding pattern of endogenous ALKBH3 in PC3 prostate cancer cells coupled with microarray experiments to examine the expression effects of ALKBH3 depletion. Results: We demonstrate that ALKBH3 binds to transcription associated locations, such as places of promoter-proximal paused RNA polymerase II and enhancers. Strikingly, ALKBH3 strongly binds to the transcription initiation sites of a small number of highly active gene promoters. These promoters are characterized by high levels of transcriptional regulators, including transcription factors, the Mediator complex, cohesin, histone modifiers, and active histone marks. Gene expression analysis showed that ALKBH3 does not directly influence the transcription of its target genes, but its depletion induces an upregulation of ALKBH3 non-bound inflammatory genes. Conclusions: The genomic binding pattern of ALKBH3 revealed a putative novel hyperactive promoter type. Further, we propose that ALKBH3 is an intrinsic DNA repair protein that suppresses transcription associated DNA damage at highly expressed genes and thereby plays a role to maintain genomic integrity in ALKBH3-overexpressing cancer cells. These results raise the possibility that ALKBH3 may be a potential target for inhibiting cancer progression. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0180-0) contains supplementary material, which is available to authorized users

Histone demethylase KDM5A is an integral part of the core Notch–RBP-J repressor complex

Timely acquisition of cell fates and the elaborate control of growth in numerous organs depend on Notch signaling. Upon ligand binding, the core transcription factor RBP-J activates transcription of Notch target genes. In the absence of signaling, RBP-J switches off target gene expression, assuring the tight spatiotemporal control of the response by a mechanism incompletely understood. Here we show that the histone demethylase KDM5A is an integral, conserved component of Notch/RBP-J gene silencing. Methylation of histone H3 Lys 4 is dynamically erased and re-established at RBP-J sites upon inhibition and reactivation of Notch signaling. KDM5A interacts physically with RBP-J; this interaction is conserved in Drosophila and is crucial for Notch-induced growth and tumorigenesis responses

The CpG Island-Binding Protein SAMD1 Contributes to an Unfavorable Gene Signature in HepG2 Hepatocellular Carcinoma Cells

The unmethylated CpG island-binding protein SAMD1 is upregulated in many human cancer types, but its cancer-related role has not yet been investigated. Here, we used the hepatocellular carcinoma cell line HepG2 as a cancer model and investigated the cellular and transcriptional roles of SAMD1 using ChIP-Seq and RNA-Seq. SAMD1 targets several thousand gene promoters, where it acts predominantly as a transcriptional repressor. HepG2 cells with SAMD1 deletion showed slightly reduced proliferation, but strongly impaired clonogenicity. This phenotype was accompanied by the decreased expression of pro-proliferative genes, including MYC target genes. Consistently, we observed a decrease in the active H3K4me2 histone mark at most promoters, irrespective of SAMD1 binding. Conversely, we noticed an increase in interferon response pathways and a gain of H3K4me2 at a subset of enhancers that were enriched for IFN-stimulated response elements (ISREs). We identified key transcription factor genes, such as IRF1, STAT2, and FOSL2, that were directly repressed by SAMD1. Moreover, SAMD1 deletion also led to the derepression of the PI3K-inhibitor PIK3IP1, contributing to diminished mTOR signaling and ribosome biogenesis pathways. Our work suggests that SAMD1 is involved in establishing a pro-proliferative setting in hepatocellular carcinoma cells. Inhibiting SAMD1’s function in liver cancer cells may therefore lead to a more favorable gene signature

Ush regulates hemocyte-specific gene expression, fatty acid metabolism and cell cycle progression and cooperates with dNuRD to orchestrate hematopoiesis.

The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator

IRF8 Is an AML-Specific Susceptibility Factor That Regulates Signaling Pathways and Proliferation of AML Cells

Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients’ prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs

Polycomb-like proteins link the PRC2 complex to CpG islands

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression1,2 and plays essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like proteins (PCLs), including PHF1, MTF2 and PHF19, are PRC2 associated factors that form sub-complexes with PRC2 core components3, and have been proposed to modulate PRC2’s enzymatic activity or its recruitment to specific genomic loci4–13. Mammalian PRC2 binding sites are enriched in CG content, which correlate with CpG islands that display a low level of DNA methylation14. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. In this study, we solved the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We found that the extended homologous (EH) regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a manner completely different from the canonical winged-helix motif-DNA recognition. We further showed that the PCL EH domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic cells. Our research provides the first direct evidence demonstrating that PCLs are critical for PRC2 recruitment to CpG islands, thereby further clarifying their roles in transcriptional regulation in vivo