Vasohibin-1 and Vasohibin-2 regulate angiogenesis, tumour growth and metastasis. Their molecular functions, however, were previously unknown, in large part owing to their perceived lack of homology ...to proteins of known structure and function. To identify their functional amino acids and domains, their molecular activity and their evolutionary history, we undertook an in-depth analysis of Vasohibin sequences. We find that Vasohibin proteins are previously undetected members of the transglutaminase-like cysteine protease superfamily, and all possess a non-canonical Cys-His-Ser catalytic triad. We further propose a calcium-dependent activation mechanism for Vasohibin proteins. These findings can now be used to design constructs for protein structure determination and to develop enzyme inhibitors as angiogenic regulators to treat metastasis and tumour growth.
luis.sanchezpulido@dpag.ox.ac.uk
Supplementary data are available at Bioinformatics online.
Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding ...autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.
The reality of pervasive transcription Clark, Michael B; Amaral, Paulo P; Schlesinger, Felix J ...
PLoS biology,
07/2011, Letnik:
9, Številka:
7
Journal Article
Recenzirano
Odprti dostop
In parallel, whole chromosome tiling array interrogation of the RNA content of a variety of human tissues and cell lines revealed that, collectively, at least 93% of genomic bases are transcribed in ...one cell type or another 1,10-13. Since it is well established that highly expressed mRNAs dominate the non-ribosomal portion of the polyA+ transcriptome 7,8,10,14-19, normalization approaches were used to reduce the quantity of highly expressed transcripts in these cDNA analyses 7,8, and are implicit in tiling array approaches. ...any estimate of the pervasiveness of transcription requires inclusion of all data sources, and less than exhaustive analyses can only provide lower bounds for transcriptional complexity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ ...that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.
The advent of next-generation sequencing, and in particular RNA-sequencing (RNA-seq), technologies has expanded our knowledge of the transcriptional capacity of human and other animal, genomes. In ...particular, recent RNA-seq studies have revealed that transcription is widespread across the mammalian genome, resulting in a large increase in the number of putative transcripts from both within, and intervening between, known protein-coding genes. Long transcripts that appear to lack protein-coding potential (long non-coding RNAs, lncRNAs) have been the focus of much recent research, in part owing to observations of their cell-type and developmental time-point restricted expression patterns. A variety of sequencing protocols are currently available for identifying lncRNAs including RNA polymerase II occupancy, chromatin state maps and − the focus of this review − deep RNA sequencing. In addition, there are numerous analytical methods available for mapping reads and assembling transcript models that predict the presence and structure of lncRNAs from RNA-seq data. Here we review current methods for identifying lncRNAs using large-scale sequencing data from RNA-seq experiments and highlight analytical considerations that are required when undertaking such projects.
Long noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo ...transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia.
•A total of 4516 lncRNAs were identified across multiple stages of B-cell development and activation.
To induce central T‐cell tolerance, medullary thymic epithelial cells (mTEC) collectively express most protein‐coding genes, thereby presenting an extensive library of tissue‐restricted antigens ...(TRAs). To resolve mTEC diversity and whether promiscuous gene expression (PGE) is stochastic or coordinated, we sequenced transcriptomes of 6,894 single mTEC, enriching for 1,795 rare cells expressing either of two TRAs, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 reproducible subpopulations representing distinct maturational trajectories, stages and subtypes, including novel mTEC subsets, such as chemokine‐expressing and ciliated TEC, which warrant further characterisation. Unexpectedly, 50 modules of genes were robustly defined each showing patterns of co‐expression within individual cells, which were mainly not explicable by chromosomal location, biological pathway or tissue specificity. Further, TSPAN8+ and GP2+ mTEC were randomly dispersed within thymic medullary islands. Consequently, these data support observations that PGE exhibits ordered co‐expression, although mechanisms underlying this instruction remain biologically indeterminate. Ordered co‐expression and random spatial distribution of a diverse range of TRAs likely enhance their presentation and encounter with passing thymocytes, while maintaining mTEC identity.
Synopsis
Single‐cell RNA sequencing reveals transcriptional heterogeneity based on both ordered and stochastic elements that enables a dedicated cell population, medullary thymic epithelial cells (mTECs), to mirror the body's full self‐antigen complement for development of central T‐cell tolerance.
mTEC are a highly heterogeneous cell population constituting distinct maturational trajectories, stages and subtypes.
Several novel mTEC subsets are identified, such as chemokineexpressing and ciliated TEC.
Promiscuous gene expression exhibits both ordered and stochastic elements of co‐expression.
Tissue restricted genes are reproducibly co‐expressed across individual mice and within mTEC maturational stages.
The mechanism underlying this order is biologically indeterminate with respect to position within the linear genome, biological pathway, or tissue specificity and tissue restricted antigens (TRAs) are spatially distributed in a random manner within the thymic medulla.
Single‐cell RNA sequencing reveals transcriptional heterogeneity based on both ordered and stochastic elements that enables a dedicated cell population to mirror the body's full self‐antigen complement for development of central T‐cell tolerance.
Although a small number of the vast array of animal long non-coding RNAs (lncRNAs) have known effects on cellular processes examined in vitro, the extent of their contributions to normal cell ...processes throughout development, differentiation and disease for the most part remains less clear. Phenotypes arising from deletion of an entire genomic locus cannot be unequivocally attributed either to the loss of the lncRNA per se or to the associated loss of other overlapping DNA regulatory elements. The distinction between cis- or trans-effects is also often problematic. We discuss the advantages and challenges associated with the current techniques for studying the in vivo function of lncRNAs in the light of different models of lncRNA molecular mechanism, and reflect on the design of experiments to mutate lncRNA loci. These considerations should assist in the further investigation of these transcriptional products of the genome.
A single change in DNA, RNA, proteins or cellular images can be useful as a biomarker of disease onset or progression. With high-throughput molecular phenotyping of single cells, it is now ...conceivable that the molecular changes occurring across thousands, or tens of thousands, of individual cells could additionally be considered as a disease biomarker. Transition to a disease state would then be reflected by the shifts in cell numbers and locations across a multidimensional space that is defined by the molecular content of cells. Realising this ambition requires a robust formulation of such a multidimensional 'cell space'
This is one of the goals of the recently launched Human Cell Atlas project. A second goal is to populate this 'cell space' with all cell types in the human body. Here, I consider the potential of the Human Cell Atlas project for improving our description and understanding of the cell-type specificity of disease.
Genomic tiling arrays, cDNA sequencing and, more recently, RNA-Seq have provided initial insights into the extent and depth of transcribed sequence across human and other genomes. These methods have ...led to greatly improved annotations of protein-coding genes, but have also identified transcription outside of annotated exons. One resultant issue that has aroused dispute is the balance of transcription of known exons against transcription outside of known exons. While non-genic ‘dark matter’ transcription was found by tiling arrays to be pervasive, it was seen to contribute only a small percentage of the polyadenylated transcriptome in some RNA-Seq experiments. This apparent contradiction has been compounded by a lack of clarity about what exactly constitutes a protein-coding gene. It remains unclear, for example, whether or not all transcripts that overlap on either strand within a genomic locus should be assigned to a single gene locus, including those that fail to share promoters, exons and splice junctions. The inability of tiling arrays and RNA-Seq to count transcripts, rather than exons or exon pairs, adds to these difficulties. While there is agreement that thousands of apparently non-coding loci are present outside of protein-coding genes in the human genome, there is vigorous debate of what constitutes evidence for their functionality. These issues will only be resolved upon the demonstration, or otherwise, that organismal or cellular phenotypes frequently result when non-coding RNA loci are disrupted.
PDF
