Rapid profiling of transcription factor-cofactor interaction networks reveals principles of epigenetic regulation.

Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-κB as a primary regulator of acutely induced H3K27ac. Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data supports clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be readily applied to different cell systems and provides a powerful approach to define TF-COF networks impacting chromatin state and gene regulation.

Plant and Soil Core Mycobiomes in a Two-Year Sorghum-Legume Intercropping System of Underutilized Crops in South Africa.

Fungal communities form close beneficial (mutualists) or detrimental (pathogens) associations with their plant hosts. Their diversity and abundance can be affected by agricultural practices which include cropping systems such as rotations and intercropping. Despite the importance of cropping systems in increasing productivity, knowledge of the fungal mycobiome and the core inhabitants for under-utilised cereal and legume crops, particularly over a period, is still limited. The core mycobiomes in plant tissues and bulk soils of a cereal-legume intercrop were characterized over two years using high-throughput sequencing. The intercropping trial consisted of sorghum, Bambara groundnut, cowpea, dry bean, and soybean. A greater number of molecular operational taxonomic units (MOTUs) were found in plant tissues compared to those from the soils and between year one and year two. Principal coordinate analyses revealed that fungal communities for each year were relatively distinct, particularly for the soils. The core mycobiome was dominated by a Davidiellaceae sp. (Cladosporium), Didymellaceae sp. 1 (Phoma), Didymellaceae sp. 2 (Epicoccum), Fusarium sp. 2, Unidentified (Ascomycota), and Cryptococcus MOTUs that were present in all plant tissues and soils of year one and two. Other key MOTUs were only specific to a year, substrate, or crop. Although the mycobiome of sorghum were more distinct than the cores of the legumes, there were still MOTUs dominant across all of the crops. Characterization of this baseline core across two years provides insight into those fungi that are always present in these crops, and that could be utilized in improving crop performance and productivity.

Baseline Data of the Fungal Phytobiome of Three Sorghum (Sorghum bicolor) Cultivars in South Africa using Targeted Environmental Sequencing.

Plant-associated fungi, or the mycobiome, inhabit plant surfaces above ground, reside in plant tissues as endophytes, or are rhizosphere in the narrow zone of soil surrounding plant roots. Studies have characterized mycobiomes of various plant species, but little is known about the sorghum mycobiome, especially in Africa, despite sorghum being one of the most important indigenous and commercial cereals in Africa. In this study, the mycobiome associated with above- and below-ground tissues of three commercial sorghum cultivars, as well as from rhizosphere and surrounding bulk soil samples, were sequenced using targeted sequencing with the Illumina MiSeq platform. Relative abundance differences between fungal communities were found between above-ground and below-ground niches, with most differences mostly in the dominant MOTUs, such as Davidiellaceae sp. (Cladosporium), Didymellaceae sp. 1 (Phoma), Fusarium, Cryptococcus and Mucor. Above-ground communities also appeared to be more diverse than below-ground communities, and plants harboured the most diversity. A considerable number of MOTUs were shared between the cultivars although, especially for NS5511, their abundances often differed. Several of the detected fungal groups include species that are plant pathogens of sorghum, such as Fusarium, and, at low levels, Alternaria and the Ustilaginomycetes. Findings from this study illustrate the usefulness of targeted sequencing of the ITS rDNA gene region (ITS2) to survey and monitor sorghum fungal communities and those from associated soils. This knowledge may provide tools for disease management and crop production and improvement.

Unmethylated Insulin DNA Is Elevated After Total Pancreatectomy With Islet Autotransplantation: Assessment of a Novel Beta Cell Marker.

Beta cell death may occur both after islet isolation and during infusion back into recipients undergoing total pancreatectomy with islet autotransplantation (TPIAT) for chronic pancreatitis. We measured the novel beta cell death marker unmethylated insulin (INS) DNA in TPIAT recipients before and immediately after islet infusion (n = 21) and again 90 days after TPIAT, concurrent with metabolic functional assessments (n = 25). As expected, INS DNA decreased after pancreatectomy (p = 0.0002). All TPIAT recipients had an elevated unmethylated INS DNA ratio in the first hours following islet infusion. In four samples (three patients), INS DNA was also assessed immediately after islet isolation and again before islet infusion to assess the impact of the isolation process: Unmethylated and methylated INS DNA fractions both increased over this interval, suggesting death of beta cells and exocrine tissue before islet infusion. Higher glucose excursion with mixed-meal tolerance testing was associated with persistently elevated INS DNA at day 90. In conclusion, we observed universal early elevations in the beta cell death marker INS DNA after TPIAT, with pronounced elevations in the islet supernatant before infusion, likely reflecting beta cell death induced by islet isolation. Persistent posttransplant elevation of INS DNA predicted greater hyperglycemia at 90 days.

Overexpression of an activated REL mutant enhances the transformed state of the human B-lymphoma BJAB cell line and alters its gene expression profile.

The human REL proto-oncogene encodes a transcription factor in the nuclear factor (NF)-kappaB family. Overexpression of REL is acutely transforming in chicken lymphoid cells, but has not been shown to transform any mammalian lymphoid cell type. In this report, we show that overexpression of a highly transforming mutant of REL (RELDeltaTAD1) increases the oncogenic properties of the human B-cell lymphoma BJAB cell line, as shown by increased colony formation in soft agar, tumor formation in SCID (severe combined immunodeficient) mice, and adhesion. BJAB-RELDeltaTAD1 cells also show decreased activation of caspase in response to doxorubicin. BJAB-RELDeltaTAD1 cells have increased levels of active nuclear REL protein as determined by immunofluorescence, subcellular fractionation and electrophoretic mobility shift assay. Overexpression of RELDeltaTAD1 in BJAB cells has transformed the gene expression profile of BJAB cells from that of a germinal center B-cell subtype of diffuse large B-cell lymphoma (DLBCL) (GCB-DLBCL) to that of an activated B-cell subtype (ABC-DLBCL), as evidenced by increased expression of many ABC-defining mRNAs. Upregulated genes in BJAB-RELDeltaTAD1 cells include several NF-kappaB targets that encode proteins previously implicated in B-cell development or oncogenesis, including BCL2, IRF4, CD40 and VCAM1. The cell system we describe here may be valuable for further characterizing the molecular details of REL-induced lymphoma in humans.

Deletion analysis and alternative splicing define a transactivation inhibitory domain in human oncoprotein REL.

Misregulation of REL, a nuclear factor-kappaB family transcription factor, has been implicated in several human lymphoid malignancies. REL has a conserved N-terminal DNA-binding/dimerization domain called the Rel homology domain (RHD) and a C-terminal transactivation domain (TAD). Here, we define the sequences (amino acids (aa) 323-422) between the RHD and TAD as a REL inhibitory domain (RID) because deletion of these sequences increases both REL transactivation and DNA binding. Furthermore, we have characterized two REL mRNA splice variants that encode proteins with alterations near RID: one lacking exon 9 sequences (aa 308-330; RELDelta9) and one with an exonized Alu fragment insertion of 32 aa after aa 307 (REL+Alu). Deletion of RID or exon 9-encoded sequences increases transactivation by GAL4-REL by approximately threefold. Moreover, deletion of RID or exon 9 sequences increases transactivation by full-length REL from certain kappaB site-containing promoters and increases DNA binding by REL. Deletion of RID does not affect REL's ability to transform chicken spleen cells. Reverse transcriptase-polymerase chain reaction analysis of mRNA from both primary lymphoma samples and several transformed tissue culture cell lines indicates that the RELDelta9 splice variant is preferentially expressed in lymphoma, suggesting that the REL transcript lacking exon 9 could serve as a marker for certain types of lymphoid tumors.

Mutation of an IKK phosphorylation site within the transactivation domain of REL in two patients with B-cell lymphoma enhances REL's in vitro transforming activity.

The human c-rel proto-oncogene (REL) encodes a subunit of the nuclear factor-kappaB (NF-kappaB) transcription factor. In this report, we have identified an identical point mutation in two human B-cell lymphomas (follicular (FL) and mediastinal) that changes serine (Ser)525 (TCA) to proline (Pro) (CCA) within the REL transactivation domain. This mutation was not identified in a similarly sized cohort of healthy individuals. In the mediastinal B-cell lymphoma, the mutation in REL is of germ-line origin. In both tumors, the S525P mutant allele is over-represented. REL-S525P shows enhanced in vitro transforming activity in chicken spleen cells. REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein shows increased expression in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 is a site for phosphorylation by IkappaB kinase (IKK) in vitro. The S525P mutation reduces IKKalpha- and tumor necrosis factor (TNF)alpha-stimulated transactivation by a GAL4-REL protein. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFalpha-induced cell death than cells transformed by wild-type REL. These results suggest that the S525P mutation contributes to the development of human B-cell lymphomas by affecting an IKKalpha-regulated transactivation activity of REL.

Introduction to NF-kappaB: players, pathways, perspectives.

This article serves as an introduction to the collection of reviews on nuclear factor-kappaB (NF-kappaB). It provides an overview of the discovery and current status of NF-kappaB as a research topic. Described are the structures, activities and regulation of the proteins in the NF-kappaB family of transcription factors. NF-kappaB signaling is primarily regulated by inhibitor kappaB (IkappaB) proteins and the IkappaB kinase complex through two major pathways: the canonical and non-canonical NF-kappaB pathways. The organization and focus of articles included in the following reviews are described, as well as likely future areas of research interest on NF-kappaB.

Mutations in the NF-kappaB signaling pathway: implications for human disease.

The nuclear factor-kappa B (NF-kappaB) signaling pathway is a multi-component pathway that regulates the expression of hundreds of genes that are involved in diverse and key cellular and organismal processes, including cell proliferation, cell survival, the cellular stress response, innate immunity and inflammation. Not surprisingly, mis-regulation of the NF-kappaB pathway, either by mutation or epigenetic mechanisms, is involved in many human and animal diseases, especially ones associated with chronic inflammation, immunodeficiency or cancer. This review describes human diseases in which mutations in the components of the core NF-kappaB signaling pathway have been implicated and discusses the molecular mechanisms by which these alterations in NF-kappaB signaling are likely to contribute to the disease pathology. These mutations can be germline or somatic and include gene amplification (e.g., REL), point mutations and deletions (REL, NFKB2, IKBA, CYLD, NEMO) and chromosomal translocations (BCL-3). In addition, human genetic diseases are briefly described wherein mutations affect protein modifiers or transducers of NF-kappaB signaling or disrupt NF-kappaB-binding sites in promoters/enhancers.

Inhibitors of NF-kappaB signaling: 785 and counting.

Nuclear factor kappa B (NF-kappaB) transcription factors regulate several important physiological processes, including inflammation and immune responses, cell growth, apoptosis, and the expression of certain viral genes. Therefore, the NF-kappaB signaling pathway has also provided a focus for pharmacological intervention, primarily in situations of chronic inflammation or in cancer, where the pathway is often constitutively active and plays a key role in the disease. Now that many of the molecular details of the NF-kappaB pathway are known, it is clear that modulators of this pathway can act at several levels. As described herein, over 750 inhibitors of the NF-kappaB pathway have been identified, including a variety of natural and synthetic molecules. These compounds include antioxidants, peptides, small RNA/DNA, microbial and viral proteins, small molecules, and engineered dominant-negative or constitutively active polypeptides. Several of these molecules act as general inhibitors of NF-kappaB induction, whereas others inhibit specific pathways of induction. In addition, some compounds appear to target multiple steps in the NF-kappaB pathway. Compounds designed as specific NF-kappaB inhibitors are not yet in clinical use, but they are likely to be developed as treatments for certain cancers and neurodegenerative and inflammatory diseases. Moreover, the therapeutic and preventative effects of many natural products may, at least in part, be due to their ability to inhibit NF-kappaB.

Good cop, bad cop: the different faces of NF-kappaB.

Complexes formed from the nuclear factor kappaB (NF-kappaB) family of transcription factors are ubiquitously expressed and are induced by a diverse array of stimuli. This results in their becoming activated in a wide variety of different settings. While the functions of NF-kappaB in many of these contexts have been the subject of intense research and are now well established, it is also clear that there is great diversity in the effects and consequences of NF-kappaB activation. NF-kappaB subunits do not necessarily regulate the same genes, in an identical manner, in all of the different circumstances in which they are induced. This review will discuss the different functions of NF-kappaB, the pathways that modulate NF-kappaB subunit activity and, in contrast to its more commonly thought of role as a promoter of cancer cell growth and survival, the ability of NF-kappaB, under some circumstances, to behave as a tumor suppressor.

Malignant transformation of primary chicken spleen cells by human transcription factor c-Rel.

Rel/NF-kappaB transcription factors control a variety of cellular processes, such as cell growth and apoptosis, that are relevant to oncogenesis, and mutations in genes encoding Rel/NF-kappaB transcription factors have been found in several human lymphoid cell cancers. In this study, we have used a sensitive cell outgrowth assay to demonstrate that wild-type human c-Rel can malignantly transform primary chicken spleen cells, and that transformation by c-Rel is accelerated by co-expression of Bc1-2. Full-length mouse c-Rel can also transform chicken spleen cells. These results are the first demonstration of a lymphoid cell malignant transforming ability for mammalian Rel/NF-kappaB transcription factors, and implicate c-Rel as a molecular target for cancer therapeutics.

The chicken RelB transcription factor has transactivation sequences and a tissue-specific expression pattern that are distinct from mammalian RelB.

Rel/NF-kappaB proteins are eukaryotic transcription factors that control the expression of genes involved in a large variety of cellular processes. Rel proteins share a highly conserved DNA-binding/dimerization domain called the Rel Homology (RH) domain. We have constructed and characterized a composite cDNA encoding most of the chicken RelB transcription factor. The predicted chicken RelB protein has a high degree of sequence similarity to other vertebrate RelB proteins within the RH domain, but is much less conserved outside this domain. Chicken RelB does not bind DNA as a homodimer, but forms DNA-binding heterodimers with NF-kappaB p50 or p52. Overexpressed chicken RelB localizes to the nucleus in chicken embryo fibroblasts, and the nonconserved C-terminal sequences of chicken RelB contain a transactivation domain that functions in chicken and mouse fibroblasts. Thus, chicken RelB has functional properties similar to other vertebrate RelB proteins. However, Western blotting of diverse chicken tissues indicates that chicken RelB is more widely expressed than mammalian RelB.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains.

Trip6 is a member of a subfamily of LIM domain proteins, including also zyxin, LPP, Ajuba, and Hic-5, which localize primarily to focal adhesion plaques. However, in this report, we demonstrate that Trip6 is largely in the nucleus in cells treated with leptomycin B, suggesting that Trip6 shuttles between nuclear and cytoplasmic compartments and that nuclear export of Trip6 is dependent on Crm1. Consistent with this finding, we have identified a nuclear export signal (NES) in Trip6, and mutation of this NES also results in sequestration of Trip6 in the nucleus. Addition of the Trip6 NES to the nuclear v-Rel oncoprotein redirects v-Rel to the cytoplasm. Trip6 also has at least two sequences that can direct cytoplasmic beta-galactosidase to the nucleus. Using GAL4 fusion proteins and reporter gene assays, we demonstrate that Trip6 has multiple transactivation domains, including one that appears to overlap with sequences of the NES. In vitro- or in vivo-synthesized Trip6, however, does not bind to DNA-cellulose. Taken together, these results are consistent with Trip6, and other members of this LIM protein family, having a role in relaying signals between focal adhesion plaques and the nucleus.

Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3.

The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappa B family. v-Rel causes rapidly fatal lymphomas in young chickens, and transforms and immortalizes chicken lymphoid cells in vitro. Several mutations that have enhanced the oncogenicity of v-Rel have been selected during in vitro and in vivo passage of v-Rel-containing retroviruses. In this report, we show that the C-terminal deletion and two point mutations (Asp-->Gly at residue 91 and Asp-->Asn at residue 437) in v-Rel make it resistant to cleavage by the cell-death protease caspase-3. In contrast, c-Rel, which has Asp residues at these sites, can be cleaved by caspase-3 in vitro as well as in vivo in cells induced to undergo apoptosis. We have characterized activities of v-Rel mutants with recreated single caspase-3 cleavage sites, two cleavage sites, or an introduced artificial cleavage site. All of these mutant v-Rel proteins are sensitive to caspase-3 cleavage in vitro, and show wild-type activity in terms of nuclear localization in chicken fibroblasts and DNA binding in vitro. Moreover, all caspase-3-sensitive v-Rel mutants transform chicken spleen cells in vitro and induce fatal lymphoid tumors in vivo to approximately the same extent as wild-type v-Rel. As with v-Rel mutants, caspase-3-resistant c-Rel mutants behave similarly to caspase-3-sensitive wild-type c-Rel in terms of DNA binding, transcriptional activation, in vitro transformation, and tumorigenicity. Mammalian c-Rel proteins can also be cleaved by caspase-3 in vitro, and a c-Rel mutant from a human pre-T lymphoma cell line is less sensitive than wild-type human c-Rel to cleavage by caspase-3. Taken together, these results demonstrate that specific mutations render oncogenic forms of Rel proteins resistant to cleavage by a cell-death caspase; however, the biological relevance of this resistance remains unclear. Nevertheless, to our knowledge, this is the first demonstration of mutations in caspase-3 recognition sites occurring during the evolution of an oncogenic protein.