Histone Methylation by NUE, a Novel Nuclear Effector of the Intracellular Pathogen Chlamydia trachomatis

Sequence analysis of the genome of the strict intracellular pathogen Chlamydia trachomatis revealed the presence of a SET domain containing protein, proteins that primarily function as histone methyltransferases. In these studies, we demonstrated secretion of this protein via a type III secretion mechanism. During infection, the protein is translocated to the host cell nucleus and associates with chromatin. We therefore named the protein nuclear effector (NUE). Expression of NUE in mammalian cells by transfection reconstituted nuclear targeting and chromatin association. In vitro methylation assays confirmed NUE is a histone methyltransferase that targets histones H2B, H3 and H4 and itself (automethylation). Mutants deficient in automethylation demonstrated diminished activity towards histones suggesting automethylation functions to enhance enzymatic activity. Thus, NUE is secreted by Chlamydia, translocates to the host cell nucleus and has enzymatic activity towards eukaryotic substrates. This work is the first description of a bacterial effector that directly targets mammalian histones

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

The MMSET Protein Is Tightly Associated with Chromatin, Mediates Methylation of Specific Histone Residues and Forms a Complex with a Histone Demethylase.

Abstract Over 40% of cases of multiple myeloma (MM) are associated with translocations of the immunoglobulin heavy (IgH) chain gene. The t(4;14) translocation, present in ca. 15% of myeloma cases, results in the overexpression of two potential oncogenes, MMSET and FGFR3, via juxtaposition of their endogenous promoters to regulatory elements of the IgH locus. The presence of t(4;14) and MMSET overexpression are adverse prognostic factors in MM irrespective of FGFR3 expression, implicating MMSET in disease pathogenesis. We previously reported the presence of repression domains and the ability of MMSET to methylate histones. Examination of a number of MMSET isoforms generated by different chromosomal breakpoints in the t(4;14) translocation indicated that a N-terminal portion of the protein containing a PWWP domain mediated tight association of MMSET with chromatin. To determine if this region of the protein could mediate DNA binding we incubated bacterially expressed MMSET with DNA cellulose. Both the N-terminal and C-terminal portions of the protein showed DNA binding activity with different affinities. To isolate DNA sequences potentially bound by MMSET we incubated immunoprecipitated MMSET with a library of human CpG island DNA fragments, selected the bound DNA and performed several rounds of re-amplification and binding of selected sequences. A small family of clones was obtained, having several sequence motifs in common, suggesting specific DNA binding activity by the MMSET complex. The C-terminal region of MMSET containing a SET domain and a PHD finger, in addition to binding naked DNA, bound to native histones H3 and H4. These data suggest that MMSET may specifically target particular genes through recognition of DNA sequences and histones, or possibly specific histone modifications. Subsequently, MMSET may regulate these genes by further modifying the adjacent chromatin. In vitro analysis showed that recombinant MMSET could methylate several lysine residues on core histones, including H3K4, H3K36 and H4K20. However, MMSET immunopurified from a t(4;14)-positive myeloma cell line was only able to methylate histone H4. Additionally, a B-cell line engineered to overexpress MMSET in a conditional manner showed a global increase in the level of tri-methylated H4K20 and modulation of specific sets of genes involved in apoptosis. To determine if MMSET could indeed affect the chromatin configuration of a model gene, MMSET was fused to the Gal4 DNA binding domain and expressed in cells harboring a chromatin-embedded Gal4 reporter. MMSET repressed this reporter and chromatin immunoprecipitation demonstrated that this was accompanied by an increase in H4K20 tri-methylation. Finally, we found that endogenous MMSET could complex with Lysine-Specific Demethylase 1 (LSD1). Accordingly, the targeting of MMSET to the Gal4 reporter gene also led to a loss of H3K4 methylation, consistent with transcriptional repression. Collectively these data indicate that MMSET is a transcriptional effector that can target specific segments of chromatin and mediate a series of repressive changes. Misexpression of MMSET may lead to significant genetic re-programming of the B cell and contribute to myeloma development.</jats:p

Determinants of sensitivity to DZNep induced apoptosis in multiple myeloma cells.

The 3-Deazaneplanocin A (DZNep), one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by quantitative RT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Ectopic expression of ALOX5 reduced sensitivity to DZNep in H929 cells. Furthermore, down-regulation of ALOX5 by RNA interference could also induce apoptosis in H929. Gene expression analysis on MM patient dataset indicated ALOX5 expression was significantly higher in MM patients compared to normal plasma cells. We also found that Bcl-2 was overexpressed in DZNep insensitive cells, and cotreatment with DZNep and ABT-737, a Bcl-2 family inhibitor, synergistically inhibited growth and induced apoptosis of DZNep insensitive MM cells. Taken together, this study shows one of mechanisms of the DZNep efficacy on MM correlates with its ability to down-regulate the ALOX5 levels. In addition, DZNep insensitivity might be associated with overexpression of Bcl-2, and the combination of ABT-737 and DZNep could synergistically induced apoptosis. These results suggest that DZNep may be exploited therapeutically for a subset of MM

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-?B pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications