OLOGRAM: Determining significance of total overlap length between genomic regions sets.

MOTIVATION: Various bioinformatics analyses provide sets of genomic coordinates of interest. Whether two such sets possess a functional relation is a frequent question. This is often determined by interpreting the statistical significance of their overlaps. However, only few existing methods consider the lengths of the overlap, and they do not provide a resolutive p-value. RESULTS: Here, we introduce OLOGRAM, which performs overlap statistics between sets of genomic regions described in BEDs or GTF. It uses Monte Carlo simulation, taking into account both the distributions of region and inter-region lengths, to fit a negative binomial model of the total overlap length. Exclusion of user-defined genomic areas during the shuffling is supported. AVAILABILITY: This tool is available through the command line interface of the pygtftk toolkit. It has been tested on Linux and OSX and is available on Bioconda and from https://github.com/dputhier/pygtftk under the GNU GPL license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Explore, edit and leverage genomic annotations using Python GTF toolkit.

MOTIVATION: While Python has become very popular in bioinformatics, a limited number of libraries exist for fast manipulation of gene coordinates in Ensembl GTF format. RESULTS: We have developed the GTF toolkit Python package (pygtftk), which aims at providing easy and powerful manipulation of gene coordinates in GTF format. For optimal performances, the core engine of pygtftk is a C dynamic library (libgtftk) while the Python API provides usability and readability for developing scripts. Based on this Python package, we have developed the gtftk command line interface that contains 57 sub-commands (v0.9.10) to ease handling of GTF files. These commands may be used to (i) perform basic tasks (e.g. selections, insertions, updates or deletions of features/keys), (ii) select genes/transcripts based on various criteria (e.g. size, exon number, transcription start site location, intron length, GO terms) or (iii) carry out more advanced operations such as coverage analyses of genomic features using bigWig files to create faceted read-coverage diagrams. In conclusion, the pygtftk package greatly simplifies the annotation of GTF files with external information while providing advance tools to perform gene analyses. AVAILABILITY AND IMPLEMENTATION: pygtftk and gtftk have been tested on Linux and MacOSX and are available from https://github.com/dputhier/pygtftk under the MIT license. The libgtftk dynamic library written in C is available from https://github.com/dputhier/libgtftk.

TCRα rearrangements identify a subgroup of NKL-deregulated adult T-ALLs associated with favorable outcome.

T-cell acute lymphoblastic leukemia (T-ALL) results from leukemic transformation of T-cell precursors arrested at specific differentiation stages, including an 'early-cortical' thymic maturation arrest characterized by expression of cytoplasmic TCRß but no surface T-cell receptor (TCR) and frequent ectopic expression of the TLX1/3 NK-like homeotic proteins (NKL). We designed a TCRα VJC PCR to identify clonal TCRα rearrangements in 32% of 127 T-ALLs, including 0/52 immature/TCRγδ lineage cases and 41/75 (55%) TCRαß lineage cases. Amongst the latter, TCRα rearrangements were not identified in 30/54 (56%) of IMß/pre-αß early-cortical T-ALLs, of which the majority (21/30) expressed TLX1/3. We reasoned that the remaining T-ALLs might express other NKL proteins, so compared transcript levels of 46 NKL in T-ALL and normal thymic subpopulations. Ectopic overexpression of 10 NKL genes, of which six are unreported in T-ALL (NKX2-3, BARHL1, BARX2, EMX2, LBX2 and MSX2), was detectable in 17/104 (16%) T-ALLs. Virtually all NKL overexpressing T-ALLs were TCRα unrearranged and ectopic NKL transcript expression strongly repressed Eα activity, suggesting that ectopic NKL expression is the major determinant in early-cortical thymic T-ALL maturation arrest. This immunogenetic T-ALL subtype, defined by TCRß VDJ but no TCRα VJ rearrangement, is associated with a favorable outcome in GRAALL-treated adult T-ALLs.

Novel CD28-responsive enhancer activated by CREB/ATF and AP-1 families in the human interleukin-2 receptor alpha-chain locus.

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) critically regulates the T-cell immune response, and the alpha chain CD25/IL-2Ralpha is required for the formation of the high-affinity receptor. Tissue-specific, inducible expression of the IL-2Ralpha gene is regulated by at least three positive regulatory regions (PRRI, PRRII, and PRRIII), but none responded to CD28 engagement in gene reporter assays although CD28 costimulation strongly amplifies IL-2Ralpha gene transcription. By DNase I hypersensitivity analysis, we have identified a novel TCR-CD3- and CD28-responsive enhancer (CD28rE) located 8.5 kb 5' of the IL-2Ralpha gene. PRRIV/CD28rE contains a functional CRE/TRE element required for CD28 signaling. The T-cell-specific, CD28-responsive expression of the IL-2Ralpha gene appears controlled through PRRIV/CD28rE by cooperation of CREB/ATF and AP-1 family transcription factors.

Chromatin remodeling by the T cell receptor (TCR)-beta gene enhancer during early T cell development: Implications for the control of TCR-beta locus recombination.

Gene targeting studies have shown that T cell receptor (TCR)-beta gene expression and recombination are inhibited after deletion of an enhancer (Ebeta) located at the 3' end of the approximately 500-kb TCR-beta locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-beta locus, the effects of Ebeta deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Ebeta contributes to major chromatin remodeling directed to an approximately 25-kb upstream domain comprised of the Dbeta-Jbeta locus regions. Accordingly, treatment of Ebeta-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-beta gene expression and promoted recombination within the Dbeta-Jbeta loci. Unexpectedly, however, epigenetic processes at distal Vbeta genes on the 5' side of the locus and at the 3' proximal Vbeta14 gene appear to be less dependent on Ebeta, suggesting that Ebeta activity is confined to a discrete region of the TCR-beta locus. These findings have implications with respect to the developmental control of TCR-beta gene recombination, and the process of allelic exclusion at this locus.

TCRalpha enhancer activation occurs via a conformational change of a pre-assembled nucleo-protein complex.

The TCR alpha enhancer (Ealpha) has served as a paradigm for studying how enhancers organize trans-activators into nucleo-protein complexes thought to recruit and synergistically stimulate the transcriptional machinery. Little is known, however, of either the extent or dynamics of Ealpha occupancy by nuclear factors during T cell development. Using dimethyl sulfate (DMS) in vivo footprinting, we demonstrate extensive Ealpha occupancy, encompassing both previously identified and novel sites, not only in T cells representing a developmental stage where Ealpha is known to be active (CD4(+)CD8(+)-DP cells), but surprisingly, also in cells at an earlier developmental stage where Ealpha is not active (CD4(-)CD8(-)-DN cells). Partial occupancy was also established in B-lymphoid but not non-lymphoid cells. In vivo DNase I footprinting, however, implied developmentally induced changes in nucleo-protein complex topography. Stage-specific differences in factor composition at Ealpha sequences were also suggested by EMSA analysis. These results, which indicate that alterations in the structure of a pre-assembled nucleo-protein complex correlate with the onset of Ealpha activity, may exemplify one mechanism by which enhancers can rapidly respond to incoming stimuli.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.