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Marcelo A Nobrega

Publications and source records attributed to Marcelo A Nobrega.

13 recordsLinked to original sources
PubMed2026

Negative regulation of human IL-33 in endothelium during allergic airway inflammation.

Lung IL-33 is involved in pathogen defense, barrier homeostasis, and development of allergic responses. We previously identified a 5 kb noncoding region within a GWAS-defined segment that regulates expression of human IL33 (hIL33) but is absent in the murine locus. To understand how this region affects IL-33 expression in vivo, we engineered 2 BAC-transgenic strains in which 166 kb of the human genome upstream of the hIL33 locus, along with a fluorescent reporter, was inserted into the murine genome, both with and without the 5 kb region. Comparison to a murine Il33 (mIl33) reporter strain revealed species-specific tropism; hIL33 reporter was mostly expressed in the endothelium, while mIl33 reporter was expressed in type 2 alveolar epithelium. hIL33 reporter expression in tracheal basal epithelium, submucosal glands, and lung microvasculature required the 5 kb region. Surprisingly, allergen and exogenous IL-33 downregulated hIL33 reporter in lung endothelium only when the 5 kb region was present. Similar IL-33-dependent downregulation of IL33 transcripts was observed in human endothelial cell lines, validating that our hIL33 reporter strain recapitulated human endothelial biology. Together, these data reveal the importance of the asthma-associated human 5 kb region in regulating human IL33 expression in a cell type- and context-dependent manner.

Interleukin-33↗
PubMed2007

Predicting tissue-specific enhancers in the human genome.

Determining how transcriptional regulatory signals are encoded in vertebrate genomes is essential for understanding the origins of multicellular complexity; yet the genetic code of vertebrate gene regulation remains poorly understood. In an attempt to elucidate this code, we synergistically combined genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to define sequence signatures characteristic of candidate tissue-specific enhancers in the human genome. We applied this strategy to microarray-based gene expression profiles from 79 human tissues and identified 7187 candidate enhancers that defined their flanking gene expression, the majority of which were located outside of known promoters. We cross-validated this method for its ability to de novo predict tissue-specific gene expression and confirmed its reliability in 57 of the 79 available human tissues, with an average precision in enhancer recognition ranging from 32% to 63% and a sensitivity of 47%. We used the sequence signatures identified by this approach to successfully assign tissue-specific predictions to approximately 328,000 human-mouse conserved noncoding elements in the human genome. By overlapping these genome-wide predictions with a data set of enhancers validated in vivo, in transgenic mice, we were able to confirm our results with a 28% sensitivity and 50% precision. These results indicate the power of combining complementary genomic data sets as an initial computational foray into a global view of tissue-specific gene regulation in vertebrates.

Animals↗
PubMed2006

In vivo enhancer analysis of human conserved non-coding sequences.

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.

Animals↗
PubMed2005

Identifying synonymous regulatory elements in vertebrate genomes.

Synonymous gene regulation, defined by regulatory elements driving shared temporal and/or spatial aspects of gene expression, is most probably predicated on genomic elements that contain similar modules of certain transcription factor binding sites (TFBS). We have developed a method to scan vertebrate genomes for evolutionary conserved modules of TFBS in a predefined configuration, and created a tool, named SynoR that identifies synonymous regulatory elements (SREs) in vertebrate genomes. SynoR performs de novo identification of SREs utilizing known patterns of TFBS in active regulatory elements (REs) as seeds for genome scans. Layers of multiple-species conservation allow the use of differential phylogenetic sequence conservation filters in search of SREs and the results are displayed such as to provide an extensive annotation of the genes containing the detected REs. Gene Ontology categories are utilized to further functionally classify the identified genes, and integrated GNF Expression Atlas 2 data allow the cataloging of tissue-specificities of the predicted SREs. SynoR is publicly available at http://synor.dcode.org.

Animals↗
PubMed2005

In vivo characterization of a vertebrate ultraconserved enhancer.

Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.

Animals↗
PubMed2004

Evolution and functional classification of vertebrate gene deserts.

Large tracts of the human genome, known as gene deserts, are devoid of protein-coding genes. Dichotomy in their level of conservation with chicken separates these regions into two distinct categories, stable and variable. The separation is not caused by differences in rates of neutral evolution but instead appears to be related to different biological functions of stable and variable gene deserts in the human genome. Gene Ontology categories of the adjacent genes are strongly biased toward transcriptional regulation and development for the stable gene deserts, and toward distinctively different functions for the variable gene deserts. Stable gene deserts resist chromosomal rearrangements and appear to harbor multiple distant regulatory elements physically linked to their neighboring genes, with the linearity of conservation invariant throughout vertebrate evolution.

Animals↗
PubMed2004

Exploiting human--fish genome comparisons for deciphering gene regulation.

Comparative genomics has served as an essential guide in the identification of functional coding and non-coding sequences in vertebrate genomes. Human-mouse pair-wise comparisons have limited utility for identifying functional conserved non-coding sequences, owing to the large number of sequences shared between these species. In searching for more stringent filters to uncover non-coding elements more likely to be of functional importance in the human genome, human-fish sequence comparisons have emerged as an important strategy, leading to the efficient identification of enhancer elements. These sequences are unevenly distributed in the genome, tending to cluster around genes involved in key developmental processes, with recent studies suggesting that they represent genomic segments in which sequence variation can result in morphological changes and innovation. These elements, conserved over long evolutionary time, emerge as primary candidates that are likely to harbor sequence variation contributing to susceptibility of human disease phenotypes.

Animals↗
PubMed2004

ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes.

With an increasing number of vertebrate genomes being sequenced in draft or finished form, unique opportunities for decoding the language of DNA sequence through comparative genome alignments have arisen. However, novel tools and strategies are required to accommodate this large volume of genomic information and to facilitate the transfer of predictions generated by comparative sequence alignment to researchers focused on experimental annotation of genome function. Here, we present the ECR Browser, a tool that provides easy and dynamic access to whole genome alignments of human, mouse, rat and fish sequences. This web-based tool (http://ecrbrowser.dcode.org) provides the starting point for discovery of novel genes, identification of distant gene regulatory elements and prediction of transcription factor binding sites. The genome alignment portal of the ECR Browser also permits fast and automated alignments of any user-submitted sequence to the genome of choice. The interconnection of the ECR Browser with other DNA sequence analysis tools creates a unique portal for studying and exploring vertebrate genomes.

Animals↗
PubMed2004

Comparative genomic analysis as a tool for biological discovery.

The recent completion of the human genome sequence has enabled the identification of a large fraction of our gene catalogue and their physical chromosomal position. However, current efforts lag at defining the cis-regulatory sequences that control the spatial and temporal patterns of each gene's expression. This task remains difficult due to our lack of knowledge of the vocabulary controlling gene regulation and the vast genomic search space, with greater than 95% of our genome being noncoding. Recent comparative genomic-based strategies are beginning to aid in the identification of functional sequences based on their high levels of evolutionary conservation. This has proven successful for comparisons between closely related species such as human-primate or human-mouse, but also holds true for distant evolutionary comparisons, such as human-fish or human-bird. In this review we provide support for the utility of cross-species sequence comparisons by illustrating several applications of this strategy, including the identification of new genes and functional non-coding sequences. We also discuss emerging concepts as this field matures, such as how to properly select which species for comparison, which may differ significantly between independent studies.

Animals↗
PubMed2004

Initial characterization of a rat model of diabetic nephropathy.

The lack of an appropriate animal model that spontaneously develops diabetic nephropathy has been a significant limitation in the search for genetic factors underlying this disease and the development of new therapeutic strategies to prevent progressive renal disease in diabetes. We introgressed the mitochondria and some passenger loci from the FHH/EurMcwi rat into the genetic background of diabetic GK rats, creating a new rat strain, T2DN (T2DN/Mcwi). Despite the high degree of genetic similarity between T2DN and GK rats (97% at 681 loci), diabetes ensues earlier and progresses more severely in T2DN rats. T2DN rats exhibit proteinuria by 6 months of age, accompanied by renal histologic abnormalities such as focal glomerulosclerosis, mesangial matrix expansion, and thickening of basement membranes. These characteristics progress over time, and nearly all T2DN rats exhibit diffuse global glomerulosclerosis with nodule formation and arteriolar hyalinosis by 18 months of age. The histologic changes in the kidney of T2DN rats closely mimic the changes seen in the kidney of patients with diabetes. These results indicate that the T2DN rat is a suitable model for investigating diabetic nephropathy. Here we report the initial genetic and physiological characterization of this new rat model of diabetic nephropathy.

Animals↗
PubMed2001

Gene expression profiling leads to identification of GLI1-binding elements in target genes and a role for multiple downstream pathways in GLI1-induced cell transformation.

The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.

Amino Acid Motifs↗