Genomic sequences exhibit self-organization properties at various hierarchical levels. One such is the gene structure of higher eukaryotes with its complex exon/intron arrangement. Exon sizes and ...exon numbers in genes have been shown to conform to a law derived from statistical linguistics and formulated by Menzerath and Altmann, according to which the mean size of the constituents of an entity is inversely related to the number of these constituents. We herein perform a detailed analysis of this property in the complete exon set of the mouse genome in correlation to the sequence conservation of each exon and the transcriptional complexity of each gene locus. We show that extensive linear fits, representative of accordance to Menzerath–Altmann law are restricted to a particular subset of genes that are formed by exons under low or intermediate sequence constraints and have a small number of alternative transcripts. Based on this observation we propose a hypothesis for the law of Menzerath–Altmann in mammalian genes being predominantly due to genes that are more versatile in function and thus, more prone to undergo changes in their structure. To this end we demonstrate one test case where gene categories of different functionality also show differences in the extent of conformity to Menzerath–Altmann law.
In primary breast cancer metastases frequently arise from a state of dormancy that may persist for extended periods of time. We investigated the efficacy of plasma micro-RNA (miR)-21, miR-23b, ...miR-190, miR-200b and miR-200c, related to dormancy and metastasis, to predict the outcome of patients with early breast cancer.
miRNAs were evaluated by RT-qPCR in plasma obtained before adjuvant chemotherapy. miRNA expression, classified as high or low according to median values, correlated with relapse and survival. Receiver operating characteristic (ROC) curves were constructed to determine miRNA sensitivity and specificity.
miR-21 (p < 0.001), miR-23b (p = 0.028) and miR-200c (p < 0.001) expression were higher and miR-190 was lower (p = 0.013) in relapsed (n = 49), compared to non-relapsed patients (n = 84). Interestingly, miR-190 was lower (p = 0.0032) in patients with early relapse (at < 3 years; n = 23) compared to those without early relapse (n = 110). On the other hand, miR-21 and miR-200c were higher (p = 0.015 and p < 0.001, respectively) in patients with late relapse (relapse at ≥ 5 years; n = 20) as compared to non-relapsed patients. High miR-200c was associated with shorter disease-free survival (DFS) (p = 0.005) and high miR-21 with both shorter DFS and overall survival (OS) (p < 0.001 and p = 0.033, respectively) compared to low expression. ROC curve analysis revealed that miR-21, miR-23b, miR-190 and miR-200c discriminated relapsed from non-relapsed patients. A combination of of miR-21, miR-23b and miR-190 showed higher sensitivity and specificity in ROC analyses compared to each miRNA alone; accuracy was further improved by adding lymph node infiltration and tumor grade to the panel of three miRs (AUC 0.873). Furthermore, the combination of miR-200c, lymph node infiltration, tumor grade and estrogen receptor predicted late relapse (AUC 0.890).
Circulating miRNAs are differentially expressed among relapsed and non-relapsed patients with early breast cancer and predict recurrence many years before its clinical detection. Our results suggest that miRNAs represent potential circulating biomarkers in early breast cancer.
Chromatin structure influences transcription, but its role in subsequent RNA processing is unclear. Here we present analyses of high-throughput data that imply a relationship between nucleosome ...positioning and exon definition. First, we have found stable nucleosome occupancy within human and Caenorhabditis elegans exons that is stronger in exons with weak splice sites. Conversely, we have found that pseudoexons--intronic sequences that are not included in mRNAs but are flanked by strong splice sites--show nucleosome depletion. Second, the ratio between nucleosome occupancy within and upstream from the exons correlates with exon-inclusion levels. Third, nucleosomes are positioned central to exons rather than proximal to splice sites. These exonic nucleosomal patterns are also observed in non-expressed genes, suggesting that nucleosome marking of exons exists in the absence of transcription. Our analysis provides a framework that contributes to the understanding of splicing on the basis of chromatin architecture.
One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about ...-1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = -1.29), intergenic regions (∆Lk = -1.23), rDNA genes (∆Lk = -1.24) and telomeric regions (∆Lk = -1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.
Mechanisms of tissue-specific gene expression regulation via 3D genome organization are poorly understood. Here we uncover the regulatory chromatin network of developing T cells and identify SATB1, a ...tissue-specific genome organizer, enriched at the anchors of promoter-enhancer loops. We have generated a T-cell specific Satb1 conditional knockout mouse which allows us to infer the molecular mechanisms responsible for the deregulation of its immune system. H3K27ac HiChIP and Hi-C experiments indicate that SATB1-dependent promoter-enhancer loops regulate expression of master regulator genes (such as Bcl6), the T cell receptor locus and adhesion molecule genes, collectively being critical for cell lineage specification and immune system homeostasis. SATB1-dependent regulatory chromatin loops represent a more refined layer of genome organization built upon a high-order scaffold provided by CTCF and other factors. Overall, our findings unravel the function of a tissue-specific factor that controls transcription programs, via spatial chromatin arrangements complementary to the chromatin structure imposed by ubiquitously expressed genome organizers.
Substantial evidence highlights divergences in immune responses between men and women. Women are more susceptible to autoimmunity, whereas men suffer from the more severe presentation of autoimmune ...disorders. The molecular mechanism of this sexual dimorphism remains elusive. Herein, we conducted a comprehensive analysis of sex differences in whole-blood gene expression focusing on alternative splicing (AS) events in systemic lupus erythematosus (SLE), which is a prototype sex-biased disease. This study included 79 SLE patients with active disease and 58 matched healthy controls who underwent whole-blood RNA sequencing. Sex differences in splicing events were widespread, existent in both SLE and a healthy state. However, we observed distinct gene sets and molecular pathways targeted by sex-dependent AS in SLE patients as compared to healthy subjects, as well as a notable sex dissimilarity in intron retention events. Sexually differential spliced genes specific to SLE patients were enriched for dynamic cellular processes including chromatin remodeling, stress and inflammatory responses. Remarkably, the extent of sexual differences in AS in the SLE patients and healthy individuals exceeded those in gene expression. Overall, this study reveals an unprecedent variation in sex-dependent splicing events in SLE and the healthy state, with potential implications for understanding the molecular basis of sexual dimorphism in autoimmunity.
Tumor progression locus-2 (Tpl2) kinase is a major inflammatory mediator in immune cell types recently found to be genetically associated with inflammatory bowel diseases (IBDs). Here we show that ...Tpl2 may exert a dominant homeostatic rather than inflammatory function in the intestine mediated specifically by subepithelial intestinal myofibroblasts (IMFs). Mice with complete or IMF-specific Tpl2 ablation are highly susceptible to epithelial injury-induced colitis showing impaired compensatory proliferation in crypts and extensive ulcerations without significant changes in inflammatory responses. Following epithelial injury, IMFs sense innate or inflammatory signals and activate, via Tpl2, the cyclooxygenase-2 (Cox-2)-prostaglandin E ₂ (PGE ₂) pathway, which we show here to be essential for the epithelial homeostatic response. Exogenous PGE ₂ administration rescues mice with complete or IMF-specific Tpl2 ablation from defects in crypt function and susceptibility to colitis. We also show that Tpl2 expression is decreased in IMFs isolated from the inflamed ileum of IBD patients indicating that Tpl2 function in IMFs may be highly relevant to human disease. The IMF-mediated mechanism we propose also involves the IBD-associated genes IL1R1 , MAPK1 , and the PGE ₂ receptor-encoding PTGER4 . Our results establish a previously unidentified myofibroblast-specific innate pathway that regulates intestinal homeostasis and may underlie IBD susceptibility in humans.
Significance Tumor progression locus-2 (Tpl2) is a proinflammatory gene genetically associated with inflammatory bowel diseases. This study provides a mechanistic interpretation for this association showing a dominant Tpl2-mediated homeostatic mechanism protecting mice from epithelial injury-induced colitis. This function of Tpl2 is mediated specifically by subepithelial intestinal myofibroblasts, a cell type supporting crypt stem cells. Tpl2 in myofibroblasts is essential for the compensatory proliferative response of the epithelium by promoting arachidonic acid metabolism and cyclooxygenase-2 (Cox-2)/prostaglandin E ₂ activation. Notably, in Crohn’s Disease patients, Tpl2 is downregulated in myofibroblasts isolated from the inflamed ileum. These results challenge current concepts on a solely proinflammatory function of Tpl2 and highlight the dominant role of subepithelial myofibroblasts in sensing inflammation and tissue damage and promoting intestinal homeostasis through Tpl2-Cox-2-prostaglandin E ₂.
Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor-suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML ...knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.
In addition to increasing the complexity of the transcriptional output, alternative RNA splicing can lead to the reduction of mRNA translation or the production of non-functional or malfunctional ...proteins, thus representing a vital component of the gene regulation process. Herein, we set out to detect and characterize alternative splicing events that occur in whole-blood samples of patients with Systemic Lupus Erythematosus (SLE) as compared to healthy counterparts. Through the implementation of a computational pipeline on published RNA-sequencing data, we identified extensive changes in the transcription dynamics affecting a large number of genes. We found a predominance of intron retention events, with the majority introducing premature stop codons, suggestive of gene repression, in both inactive and active SLE patient samples. Alternative splicing affected a distinct set of genes from the ones detected as differentially expressed in the same comparisons, while alternatively spliced genes tended to reside in genome areas associated with increased gene co-expression. Functional analysis of genes affected by alternative splicing pointed towards particular functions related to metabolism and histone acetylation as of potential interest. Together, our findings underline the importance of incorporating alternative splicing analyses in the context of molecular characterization of complex diseases such as SLE.
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