Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5′-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes

Agrin Binds BMP2, BMP4 and TGFβ1

The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFβs, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFβ family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFβ1 with relatively high affinity, the KD values of the interactions calculated from SPR experiments fall in the 10−8 M–10−7 M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at ∼5×10−7 M. Paradoxically, in the case of TGFβ1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFβ family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFβ family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering

EuroPineDB: a high-coverage web database for maritime pine transcriptome

Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network

Long non-coding RNA RAMS11 promotes metastatic colorectal cancer progression

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC

Towards the fast scrambling conjecture

Many proposed quantum mechanical models of black holes include highly nonlocal interactions. The time required for thermalization to occur in such models should reflect the relaxation times associated with classical black holes in general relativity. Moreover, the time required for a particularly strong form of thermalization to occur, sometimes known as scrambling, determines the time scale on which black holes should start to release information. It has been conjectured that black holes scramble in a time logarithmic in their entropy, and that no system in nature can scramble faster. In this article, we address the conjecture from two directions. First, we exhibit two examples of systems that do indeed scramble in logarithmic time: Brownian quantum circuits and the antiferromagnetic Ising model on a sparse random graph. Unfortunately, both fail to be truly ideal fast scramblers for reasons we discuss. Second, we use Lieb-Robinson techniques to prove a logarithmic lower bound on the scrambling time of systems with finite norm terms in their Hamiltonian. The bound holds in spite of any nonlocal structure in the Hamiltonian, which might permit every degree of freedom to interact directly with every other one.Comment: 34 pages. v2: typo correcte

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development

Predicting Human Nucleosome Occupancy from Primary Sequence

Nucleosomes are the fundamental repeating unit of chromatin and comprise the structural building blocks of the living eukaryotic genome. Micrococcal nuclease (MNase) has long been used to delineate nucleosomal organization. Microarray-based nucleosome mapping experiments in yeast chromatin have revealed regularly-spaced translational phasing of nucleosomes. These data have been used to train computational models of sequence-directed nuclesosome positioning, which have identified ubiquitous strong intrinsic nucleosome positioning signals. Here, we successfully apply this approach to nucleosome positioning experiments from human chromatin. The predictions made by the human-trained and yeast-trained models are strongly correlated, suggesting a shared mechanism for sequence-based determination of nucleosome occupancy. In addition, we observed striking complementarity between classifiers trained on experimental data from weakly versus heavily digested MNase samples. In the former case, the resulting model accurately identifies nucleosome-forming sequences; in the latter, the classifier excels at identifying nucleosome-free regions. Using this model we are able to identify several characteristics of nucleosome-forming and nucleosome-disfavoring sequences. First, by combining results from each classifier applied de novo across the human ENCODE regions, the classifier reveals distinct sequence composition and periodicity features of nucleosome-forming and nucleosome-disfavoring sequences. Short runs of dinucleotide repeat appear as a hallmark of nucleosome-disfavoring sequences, while nucleosome-forming sequences contain short periodic runs of GC base pairs. Second, we show that nucleosome phasing is most frequently predicted flanking nucleosome-free regions. The results suggest that the major mechanism of nucleosome positioning in vivo is boundary-event-driven and affirm the classical statistical positioning theory of nucleosome organization

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

<p>Abstract</p> <p>Background</p> <p>Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by <it>ERBB2</it> (<it>HER-2/neu</it>) oncogene expression.</p> <p>Results</p> <p>The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of <it>ERBB2</it>-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of <it>ERBB2</it>. The relative expression balance between AS variants from 3 genes was differentially modulated by <it>ERBB2</it> in this model system.</p> <p>Conclusions</p> <p>In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by <it>ERBB2</it>-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.</p