Stable Transcription Research Articles

Mycobacterial RNA Polymerase Requires a U-Tract at Intrinsic Terminators and Is Aided by NusG at Suboptimal Terminators

ABSTRACTIntrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich “U-tract,” play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently.

EPA-1718 - Neuropathological alterations in FTD cases with C9ORF72 mutation - new insights

A G4C2 repeat extension in the first intron of C9ORF72 is the most common cause of familial frontotemporal dementia with and without motoneuron disease or atypical Parkinsonism. We recently found that the characteristic p62 positive/TDP43 negative neuronal cytoplasmic inclusions (NCIs) mainly seen in cerebellum und hippocampus consist of different dipeptide repeat proteins (DPRs) generated by an ATG independent translation of stable sense and antisense transcripts of the extended intron. After creating specific antibodies against all potential DPRs resulting from different reading frames, we investigated their regional and cellular distribution pattern in the central nervous system of autopsy cases with C9ORF72 mutation by immunohistochemistry. Aggregates of all DPRs were seen in neuronal cell bodies and processes. Glycine-alanine and glycine-prolin DPRs dominated. NCIs were abundant in all neocortical areas, in the hippocampal formation and in cerebellum, less frequent in subcortical nuclei, and rare in brain stem and spinal cord following a rostro-caudal gradient. Different DPRs were found in the same NCI. The regional distribution pattern of NCIs was similar in all clinical subtypes, and did not directly correlate with neurodegeneration. DPRs and TDP43 that usually also aggregates in C9ORF72 mutation cases were rarely co-localized in the same NCI. In case of co-localization DPR proteins formed a central core surrounded by TDP43. The detection of DPR inclusions directly connects the mutation with specific neuropathological alterations. The formation of DPR inclusions seems to precede the formation of TDP43 inclusions. If there is a neurotoxic effect of DPRs, DPR inclusions might be neuroprotective.

Androgen receptor complexes probe DNA for recognition sequences by short random interactions

Owing to the tremendous progress in microscopic imaging of fluorescently labeled proteins in living cells, the insight into the highly dynamic behavior of transcription factors has rapidly increased over the past decade. However, a consistent quantitative scheme of their action is still lacking. Using the androgen receptor (AR) as a model system, we combined three different fluorescence microscopy assays: single-molecule microscopy, photobleaching and correlation spectroscopy, to provide a quantitative model of the action of this transcription factor. This approach enabled us to distinguish two types of AR-DNA binding: very brief interactions, in the order of a few hundred milliseconds, and hormone-induced longer-lasting interactions, with a characteristic binding time of several seconds. In addition, freely mobile ARs were slowed down in the presence of hormone, suggesting the formation of large AR-co-regulator complexes in the nucleoplasm upon hormone activation. Our data suggest a model in which mobile hormone-induced complexes of transcription factors and co-regulators probe DNA by briefly binding at random sites, only forming relatively stable transcription initiation complexes when bound to specific recognition sequences.

Biochemical Characterization of Patients With In-Frame or Out-of-FrameDMDDeletions Pertinent to Exon 44 or 45 Skipping

In Duchenne muscular dystrophy (DMD), the reading frame of an out-of-frame DMD deletion can be repaired by antisense oligonucleotide (AO)-mediated exon skipping. This creates a shorter dystrophin protein, similar to those expressed in the milder Becker muscular dystrophy (BMD). The skipping of some exons may be more efficacious than others. Patients with exon 44 or 45 skippable deletions (AOs in clinical development) have a less predictable phenotype than those skippable for exon 51, a group in advanced clinical trials. A way to predict the potential of AOs is the study of patients with BMD who have deletions that naturally mimic those that would be achieved by exon skipping. To quantify dystrophin messenger RNA (mRNA) and protein expression in patients with DMD deletions treatable by, or mimicking, exon 44 or 45 skipping. Retrospective study of nondystrophic controls (n = 2), patients with DMD (n = 5), patients with intermediate muscular dystrophy (n = 3), and patients with BMD (n = 13) at 4 university-based academic centers and pediatric hospitals. Biochemical analysis of existing muscle biopsies was correlated with the severity of the skeletal muscle phenotype. Dystrophin mRNA and protein expression. Patients with DMD who have out-of-frame deletions skippable for exon 44 or 45 had an elevated number of revertant and trace dystrophin expression (approximately 19% of control, using quantitative immunohistochemistry) with 4 of 9 patients presenting with an intermediate muscular dystrophy phenotype (3 patients) or a BMD-like phenotype (1 patient). Corresponding in-frame deletions presented with predominantly mild BMD phenotypes and lower dystrophin levels (approximately 42% of control) than patients with BMD modeling exon 51 skipping (approximately 80% of control). All 12 patients with in-frame deletions had a stable transcript compared with 2 of 9 patients with out-of-frame deletions (who had intermediate muscular dystrophy and BMD phenotypes). Exon 44 or 45 skipping will likely yield lower levels of dystrophin than exon 51 skipping, although the resulting protein is functional enough to often maintain a mild BMD phenotype. Dystrophin transcript stability is an important indicator of dystrophin expression, and transcript instability in DMD compared with BMD should be explored as a potential biomarker of response to AOs. This study is beneficial for the planning, execution, and analysis of clinical trials for exon 44 and 45 skipping.

The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

Integrative Analysis of mRNA Expression and Half-Life Data Reveals Trans-Acting Genetic Variants Associated with Increased Expression of Stable Transcripts

Genetic variation in gene expression makes an important contribution to phenotypic variation and susceptibility to disease. Recently, a subset of cis-acting expression quantitative loci (eQTLs) has been found to result from polymorphisms that affect RNA stability.Here we carried out a search for trans-acting variants that influence RNA stability. We first demonstrate that differences in the activity of trans-acting factors that stabilize RNA can be detected by comparing the expression levels of long-lived (stable) and short-lived (unstable) transcripts in high-throughput gene expression experiments. Using gene expression microarray data generated from eight HapMap3 populations, we calculated the relative expression ranks of long-lived transcripts versus short-lived transcripts in each sample. Treating this as a quantitative trait, we applied genome-wide association and identified a single nucleotide polymorphism (SNP), rs6137010, on chromosome 20p13 with which it is strongly associated in two Asian populations (p = 4×10−10 in CHB – Han Chinese from Beijing; p = 1×10−4 in JPT – Japanese from Tokyo). This SNP is a cis-eQTL for SNRPB in CHB and JPT but not in the other six HapMap3 populations. SNRPB is a core component of the spliceosome, and has previously been shown to affect the expression of many RNA processing factors.We propose that a cis-eQTL of SNRPB may be directly responsible for inter-individual variation in relative expression of long-lived versus short-lived transcript in Asian populations. In support of this hypothesis, knockdown of SNRPB results in a significant reduction in the relative expression of long-lived versus short-lived transcripts. Samples with higher relative expression of long-lived transcripts also had higher relative expression of coding compared to non-coding RNA and of RNA from housekeeping compared to non-housekeeping genes, due to the lower decay rates of coding RNAs, particularly those that perform housekeeping functions, compared to non-coding RNAs.

A 1-bp duplication in TGFB2 in three family members with a syndromic form of thoracic aortic aneurysm

A number of autosomal dominantly inherited disorders, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), are associated with predisposition to thoracic aortic aneurysms and dissections (TAADs). In the majority of cases, mutations in genes encoding components of the transforming growth factor-β (TGF-β) signaling pathway, such as FBN1, TGFBR1, TGFBR2 and SMAD3, underlie the disease. Recently, a familial syndromic form of TAAD with other clinical features that overlap the MFS-LDS spectrum has been described to be caused by heterozygous loss-of-function mutations in TGFB2, encoding the TGF-β2 ligand of TGF-β serine/threonine kinase receptors (TGFBRs). We analyzed the TGFB2 gene by sequencing in a cohort of 88 individuals with a Marfan-like phenotype and/or TAAD, who did not have mutations in known genes causing thoracic aortic disease. We identified the novel heterozygous c.1165dupA mutation in exon 7 of TGFB2 in three members of a family, a 51-year-old male, his brother and nephew with aortic aneurysms, cervical arterial tortuosity and/or skeletal abnormalities as well as craniofacial dysmorphisms. The 1-bp duplication causes a frameshift leading to a stable transcript with a premature stop codon after seven TGF-β2-unrelated amino acids (p.Ser389Lysfs*8). As the resulting protein is unlikely functional and by considering data from the literature, we support the notion that functional haploinsufficiency for TGF-β2 predisposes to thoracic aortic disease. Taken together, TGFB2 is a rarely mutated gene in patients with syndromic TAAD, and the clinical features of our TGFB2 mutation-positive individuals fit in the scheme of LDS, rather than MFS-related disorders.

The Human Nuclear Poly(A)-Binding Protein Promotes RNA Hyperadenylation and Decay

Control of nuclear RNA stability is essential for proper gene expression, but the mechanisms governing RNA degradation in mammalian nuclei are poorly defined. In this study, we uncover a mammalian RNA decay pathway that depends on the nuclear poly(A)-binding protein (PABPN1), the poly(A) polymerases (PAPs), PAPα and PAPγ, and the exosome subunits RRP6 and DIS3. Using a targeted knockdown approach and nuclear RNA reporters, we show that PABPN1 and PAPα, redundantly with PAPγ, generate hyperadenylated decay substrates that are recognized by the exosome and degraded. Poly(A) tail extension appears to be necessary for decay, as cordycepin treatment or point mutations in the PAP-stimulating domain of PABPN1 leads to the accumulation of stable transcripts with shorter poly(A) tails than controls. Mechanistically, these data suggest that PABPN1-dependent promotion of PAP activity can stimulate nuclear RNA decay. Importantly, efficiently exported RNAs are unaffected by this decay pathway, supporting an mRNA quality control function for this pathway. Finally, analyses of both bulk poly(A) tails and specific endogenous transcripts reveals that a subset of nuclear RNAs are hyperadenylated in a PABPN1-dependent fashion, and this hyperadenylation can be either uncoupled or coupled with decay. Our results highlight a complex relationship between PABPN1, PAPα/γ, and nuclear RNA decay, and we suggest that these activities may play broader roles in the regulation of human gene expression.

ΔFosB induction correlates inversely with CB1 receptor desensitization in a brain region-dependent manner following repeated Δ9-THC administration

Repeated Δ9-tetrahydrocannabinol (THC) administration produces desensitization and downregulation of cannabinoid type 1 receptors (CB1Rs) in the brain, but the magnitude of these adaptations varies among regions. CB1Rs in the striatum and its output regions exhibit the least magnitude and slowest development of desensitization and downregulation. The molecular mechanisms that confer these region-dependent differences are not known. The stable transcription factor, ΔFosB, is induced in the striatum following repeated THC administration and could regulate CB1Rs. To directly compare the regional profile of ΔFosB induction and CB1R desensitization and downregulation, mice were treated with THC (10 mg/kg) or vehicle for 13.5 days. CP55,940-stimulated [35S]GTPγS autoradiography and immunohistochemistry were performed to measure CB1R desensitization and downregulation, respectively, and ΔFosB expression was measured by immunoblot. Significant CB1R desensitization and downregulation occurred in the prefrontal cortex, lateral amygdala and hippocampus; desensitization was found in the basomedial amygdala and no changes were seen in remaining regions. ΔFosB was induced in the prefrontal cortex, caudate-putamen, nucleus accumbens and lateral amygdala. An inverse regional relationship between ΔFosB expression and CB1R desensitization was found, such that regions with the greatest ΔFosB induction did not exhibit CB1R desensitization and areas without ΔFosB induction had the greatest desensitization, with remaining regions exhibiting intermediate levels of both. Dual immunohistochemistry in the striatum showed both CB1R co-localization with ΔFosB in cells and CB1R puncta surrounding ΔFosB-positive cells. THC-induced expression of ΔFosB was absent in the striatum of CB1R knockout mice. These data suggest that transcriptional targets of ΔFosB might inhibit CB1R desensitization and/or that ΔFosB induction could be limited by CB1R desensitization.

Validation of reference transcripts in strawberry (Fragaria spp.)

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

Overexpression of PtSOS2 Enhances Salt Tolerance in Transgenic Poplars

Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we cloned and identified three salt overly sensitive 2 (SOS2) genes in the woody plant Populus trichocarpa, designated as PtSOS2.1, PtSOS2.2, and PtSOS2.3, which were transformed into hybrid poplar clone T89 (Populus tremula× Populus tremuloides Michx clone T89) mediated by Agrobacterium tumefaciens. Southern and northern blot analyses verified that the three genes integrated into the plant genome, and were expressed at a stable transcription level. Meanwhile, overexpression of all three PtSOS2 genes did not retard the growth of plants under normal conditions. Instead, it promoted growth in both agar-medium and soil conditions in response to salinity stress. Under salt stress, overexpression of PtSOS2.1, PtSOS2.2, and PtSOS2.3 increased the concentrations of proline and photosynthetic pigments, and the relative water content (RWC), and the activity of antioxidant enzymes, and decreased the malondialdehyde (MDA) concentrations in transgenic lines compared to the control. These results suggest that overexpression of PtSOS2 plays a significant role in improving the salt tolerance of poplars, reducing the damage to membrane structures, and enhancing osmotic adjustment and antioxidative enzyme regulation under salt stress.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-013-0640-x) contains supplementary material, which is available to authorized users.

Construction of a 5′-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis

Different mRNA stabilizing elements including the 3′-stem-loop, the ribosome binding sites (RBS), the 5′-stem-loop and the spacer region between the RBS and the 5′-stem-loop were analysed in detail to increase mRNA stability resulting in enhanced expression of heterologous proteins. In addition, in combination with mRNA stabilizing elements, we propose a new class of 5′-mRNA controllable stabilizing element (CoSE) which is composed of a transcriptional operator such as lacO of the Escherichia coli lac operon and a suitable RBS followed by an optimal spacer length. Such a CoSE allowed Bacillus subtilis cells to synthesize extraordinary stable transcripts with a half-life of the model bgaB reporter transcript (codes for an β-galactosidase gene derived from Bacillus stearothermophilus) of more than 60min. This CoSE will be an important tool to control mRNA stability in cells for both research and biotechnological applications. For example, this CoSE can be used in inducible expression vectors offering two major advantages: (i) controlling transcription of target genes by the inducer and (ii) enhancing the stability of the transcript allowing the production high levels of recombinant proteins.

The synthesis of proteins in unnucleated blood platelets

Platelets are the smallest, unnucleated blood cells that play a key role in maintaining normal hemostasis. In the human body about 1x1011 platelets are formed every day, as a the result of complex processes of differentiation, maturation and fragmentation of megakaryocytes. Studies done over 4 decades ago demonstrated that circulating in blood mature platelets can synthesize proteins. Recent discoveries confirm protein synthesis by unnucleated platelets in response to activation. Moreover, protein synthesis alters the phenotype and function of platelets. Platelets synthesize several proteins involved in hemostasis (COX, αIIbβ3, TF PAI-1, Factor XI, protein C inhibitor) and in inflammatory process (IL-1β, CCL5/RANTES). In spite of lack of transcription platelets have a stable mRNA transcripts with a long life correlated with platelet life span. Platelets also show expression of two important key regulators of translation eIF4E and EIF-2α and have a variety of miRNA molecules responsible for translational regulation. This article describes the historical overview of research on protein synthesis by platelets and presents the molecular mechanisms of protein synthesis in activated platelets (and synthesis of the most important platelet proteins).

Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling

Different semen storage and sperm purification methods may affect the integrity of isolated spermatozoal RNA. RNA-Seq was applied to determine whether semen storage methods (pelleted vs. liquefied) and somatic cell lysis buffer (SCLB) vs. PureSperm (PS) purification methods affect the quantity and quality of sperm RNA. The results indicate that the method of semen storage does not markedly impact RNA profiling whereas the choice of purification can yield significant differences. RNA-Seq showed that the majority of mitochondrial and mid-piece associated transcripts were lost after SCLB purification, which indicated that the mid-piece of spermatozoa may have been compromised. In addition, the number of stable transcript pairs from SCLB-samples was less than that from the PS samples. This study supports the view that PS purification better maintains the integrity of spermatozoal RNAs.

Dismantling Promoter-driven RNA Polymerase II Transcription Complexes in Vitro by the Termination Factor Rat1

Proper RNA polymerase II (Pol II) transcription termination is essential to generate stable transcripts, to prevent interference at downstream loci, and to recycle Pol II back to the promoter (1-3). As such, termination is an intricately controlled process that is tightly regulated by a variety of different cis- and trans-acting factors (4, 5). Although many eukaryotic termination factors have been identified to date, the details of the precise molecular mechanisms governing termination remain to be elucidated. We devised an in vitro transcription system to study specific Pol II termination. We show for the first time that the exonucleolytic Rat1·Rai1 complex can elicit the release of stalled Pol II in vitro and can do so in the absence of other factors. We also find that Rtt103, which interacts with the Pol II C-terminal domain (CTD) and with Rat1, can rescue termination activity of an exonucleolytically deficient Rat1 mutant. In light of our findings, we posit a model whereby functional nucleolytic activity is not the feature of Rat1 that ultimately promotes termination. Degradation of the nascent transcript allows Rat1 to pursue Pol II in a guided fashion and arrive at the site of RNA exit from Pol II. Upon this arrival, however, it is perhaps the specific and direct contact between Rat1 and Pol II that transmits the signal to terminate transcription.

A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression1. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia (AML), is a transcription factor implicated in both gene repression and activation2. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis3–6, making it important to identify coregulatory factors that “read” the NHR2 oligomerization and contribute to leukaemogenesis4. We now show that, in leukaemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO–induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO and a potential therapeutic target in t(8;21)+ AML.

The Candidate Histocompatibility Locus of a Basal Chordate Encodes Two Highly Polymorphic Proteins

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid–secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5′ terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Abstract LB-111: A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis.

Abstract Transcription factors are frequently altered in leukemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukemia (AML), is a transcription factor implicated in both gene repression and activation. AML1-ETO exists as dimer/oligomer through its NHR2 dimerization domain. Given that the AML1-ETO dimerization has been shown to be critical for its leukemogenic activity, it is important to identify coregulatory factors that “read” the NHR2 dimerization and contribute to leukemogenesis. We now show that, in leukemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several hematopoietic transcription factors and cofactors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukemogenesis. Within the AETFC complex, the AML1-ETO dimerization is required for a specific interaction between the dimerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic study of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric transcription factors create a new protein-binding interface through dimerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced hematopoietic stem cell self-renewal and leukemogenesis. These results reveal new mechanisms of action of AML1-ETO in leukemogenesis and provide potential therapeutic possibilities for targeting t(8;21)+ AML. Citation Format: Xiao-Jian Sun, Zhanxin Wang, Lan Wang, Yanwen Jiang, T. David Soong, Nils Kost, Wei-Yi Chen, Olivier Elemento, Wolfgang Fischle, Ari Melnick, Dinshaw J. Patel, Stephen D. Nimer, Robert G. Roeder. A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2013-LB-111

Inhibition of Viral Pathogenesis and Promotion of the Septic Shock Response to Bacterial Infection by IRF-3 Are Regulated by the Acetylation and Phosphorylation of Its Coactivators

ABSTRACTInterferon (IFN) is required for protecting mice from viral pathogenesis; reciprocally, it mediates the deleterious septic shock response to bacterial infection. The critical transcription factor for IFN induction, in both cases, is IRF-3, which is activated by TLR3 or RIG-I signaling in response to virus infection and TLR4 signaling in response to bacterial infection. Here, we report that IRF-3’s transcriptional activity required its coactivators, β-catenin and CBP, to be modified by HDAC6-mediated deacetylation and protein kinase C isozyme β (PKC-β)-mediated phosphorylation, respectively, so that activated nuclear IRF-3 could form a stable transcription initiation complex at the target gene promoters. β-Catenin bridges IRF-3 and CBP, and the modifications were required specifically for the interaction between β-catenin and CBP but not β-catenin and IRF-3. Consequently, like IRF-3−/− mice, HDAC6−/− mice were resistant to bacterial lipopolysaccharide-induced septic shock. Conversely, they were highly susceptible to pathogenesis caused by Sendai virus infection. Thus, HDAC6 is an essential component of the innate immune response to microbial infection.