Structural Biology of Human H3K9 Methyltransferases

SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Enhanced version: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available i

Regulation of Tumor Suppressor p53 and HCT116 Cell Physiology by Histone Demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53−/− HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53−/− compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway

A homogeneous method for investigation of methylation-dependent protein-protein interactions in epigenetics.

Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1beta and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions

Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumor cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function