Elena Prosperini

EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer

Recent experiments have demonstrated that the Polycomb group (PcG) gene EZH2 is highly expressed in metastatic prostate cancer and in lymphomas. EZH2 is a component of the PRC2 histone methyltransferase complex, which also contains EED and SUZ12 and is required for the silencing of HOX gene expression during embryonic development. Here we demonstrate that both EZH2 and EED are essential for the proliferation of both transformed and non‐transformed human cells. In addition, the pRB‐E2F pathway tightly regulates their expression and, consistent with this, we find that EZH2 is highly expressed in a large set of human tumors. These results raise the question whether EZH2 is a marker of proliferation or if it is actually contributing to tumor formation. Significantly, we propose that EZH2 is a bona fide oncogene, since we find that ectopic expression of EZH2 is capable of providing a proliferative advantage to primary cells and, in addition, its gene locus is specifically amplified in several primary tumors.

The Histone H3 Lysine-27 Demethylase Jmjd3 Links Inflammation to Inhibition of Polycomb-Mediated Gene Silencing

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.

Latent Enhancers Activated by Stimulation in Differentiated Cells

According to current models, once the cell has reached terminal differentiation, the enhancer repertoire is completely established and maintained by cooperatively acting lineage-specific transcription factors (TFs). TFs activated by extracellular stimuli operate within this predetermined repertoire, landing close to where master regulators are constitutively bound. Here, we describe latent enhancers, defined as regions of the genome that in terminally differentiated cells are unbound by TFs and lack the histone marks characteristic of enhancers but acquire these features in response to stimulation. Macrophage stimulation caused sequential binding of stimulus-activated and lineage-determining TFs to these regions, enabling deposition of enhancer marks. Once unveiled, many of these enhancers did not return to a latent state when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents.

CDC25A PHOSPHATASE IS A TARGET OF E2F AND IS REQUIRED FOR EFFICIENT E2F-INDUCED S PHASE

ABSTRACT Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G0-G1 phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.

The Histone H3 Lysine 27-Specific Demethylase Jmjd3 Is Required for Neural Commitment

Patterns of methylation at lysine 4 and 27 of histone H3 have been associated with states of gene activation and repression that are developmentally regulated and are thought to underlie the establishment of lineage specific gene expression programs. Recent studies have provided fundamental insight into the problem of lineage specification by comparing global changes in chromatin and transcription between ES and neural stem (NS) cells, points respectively of departure and arrival for neural commitment. With these maps of the differentiated state in place, a central task is now to unravel the chromatin dynamics that enables these differentiation transitions. In particular, the observation that lineage-specific genes repressed in ES cells by Polycomb-mediated H3-K27 trimethylation (H3-K27me3) are demethylated and derepressed in differentiated cells posited the existence of a specific H3-K27 demethylase. In order to gain insight into the epigenetic transitions that enable lineage specification, we investigated the early stages of neural commitment using as model system the monolayer differentiation of mouse ES cells into neural stem (NS) cells. Starting from a comprehensive profiling of JmjC-domain genes, we report here that Jmjd3, recently identified as a H3-K27me3 specific demethylase, controls the expression of key regulators and markers of neurogenesis and is required for commitment to the neural lineage. Our results demonstrate the relevance of an enzymatic activity that antagonizes Polycomb regulation and highlight different modalities through which the dynamics of H3-K27me3 is related to transcriptional output. By showing that the H3-K27 demethylase Jmjd3 is required for commitment to the neural lineage and that it resolves the bivalent domain at the Nestin promoter, our work confirms the functional relevance of bivalent domain resolution that had been posited on the basis of the genome-wide correlation between their controlled resolution and differentiation. In addition, our data indicate that the regulation of H3-K27me3 is highly gene- and context- specific, suggesting that the interplay of methyltransferases and demethylases enables the fine-tuning more than the on/off alternation of methylation states.

Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages

Histone deacetylases (HDACs) regulate inflammatory gene expression, as indicated by the potent antiinflammatory activity of pan-HDAC inhibitors. However, the specific contribution of each of the 11 HDAC proteins to the inflammatory gene expression program is unknown. Using an integrated genomic approach, we found that Hdac3-deficient macrophages were unable to activate almost half of the inflammatory gene expression program when stimulated with LPS. A large part of the activation defect was attributable to loss of basal and LPS-inducible expression of IFN-β, which maintains Stat1 protein levels in unstimulated cells and acts in an autocrine/paracrine manner after stimulation to promote a secondary wave of Stat1-dependent gene expression. Loss of Hdac3-mediated repression of nuclear receptors led to hyperacetylation of thousands of genomic sites and associated gene derepression. The up-regulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), a nuclear receptor target, had a causative role in the phenotype because its chemical inhibition reverted, albeit partially, the Ifn-β activation defect. These data indicate a central role for Hdac3 in inflammation and may have relevance for the use of selective Hdac inhibitors as antiinflammatory agents.

Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization. A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild‐type protein. We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20. We propose a model that features the kinetochore as a ‘folding factory’ for the formation of a Mad2–Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.

ATAD2 Is a Novel Cofactor for MYC, Overexpressed and Amplified in Aggressive Tumors

The E2F and MYC transcription factors are critical regulators of cell proliferation and contribute to the development of human cancers. Here, we report on the identification of a novel E2F target gene, ATAD2, the predicted protein product of which contains both a bromodomain and an ATPase domain. The pRB-E2F pathway regulates ATAD2 expression, which is limiting for the entry into the S phase of the cell cycle. We show that ATAD2 binds the MYC oncogene and stimulates its transcriptional activity. ATAD2 maps to chromosome 8q24, 4.3 Mb distal to MYC, in a region that is frequently found amplified in cancer. Consistent with this, we show that ATAD2 expression is high in several human tumors and that the expression levels correlate with clinical outcome of breast cancer patients. We suggest that ATAD2 links the E2F and MYC pathways and contributes to the development of aggressive cancer through the enhancement of MYC-dependent transcription.

A Novel Repressive E2F6 Complex Containing the Polycomb Group Protein, EPC1, That Interacts with EZH2 in a Proliferation-specific Manner

The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA. Whereas one complex is involved in the repression of classic E2F target genes in G0, a role for E2F6 within the cell cycle has yet to be defined. We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1. We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity. Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein. Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines. EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells. Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.

The Histone Methyltransferase Wbp7 Controls Macrophage Function through GPI Glycolipid Anchor Synthesis

Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1-MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7(-/-) macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS) and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate a specialized biological role for Wbp7 in macrophage function and antimicrobial response.