Rui Pires Martins

Short communicationGlobal functional profiling of gene expression

The typical result of a microarray experiment is a list of tens or hundreds of genes found to be differentially regulated in the condition under study. Independent of the methods used to select these genes, the common task faced by any researcher is to translate these lists of genes into a better understanding of the biological phenomena involved. Currently, this is done through a tedious combination of searches through the literature and a number of public databases. We developed Onto-Express (OE) as a novel tool able to automatically translate such lists of differentially regulated genes into functional profiles characterizing the impact of the condition studied. OE constructs functional profiles (using Gene Ontology terms) for the following categories: biochemical function, biological process, cellular role, cellular component, molecular function, and chromosome location. Statistical significance values are calculated for each category. We demonstrate the validity and the utility of this comprehensive global analysis of gene function by analyzing two breast cancer datasets from two separate laboratories. OE was able to identify correctly all biological processes postulated by the original authors, as well as discover novel relevant mechanisms.

Decondensing the protamine domain for transcription.

Potentiation is the transition from higher-order, transcriptionally silent chromatin to a less condensed state requisite to accommodating the molecular elements required for transcription. To examine the underlying mechanism of potentiation an ≈13.7-kb mouse protamine domain of increased nuclease sensitivity flanked by 5′ and 3′ nuclear matrix attachment regions was defined. The potentiated DNase I-sensitive region is formed at the pachytene spermatocyte stage with the recruitment to the nuclear matrix of a large ≈9.6-kb region just upstream of the domain. Attachment is then specified in the transcribing round spermatid, recapitulating the organization of the human cluster. In comparison to other modifiers that have no effect, i.e., histone methylation, HP1, and SATB1, topoisomerase engages nuclear matrix binding as minor marks of histone acetylation appear. Reorganization is marked by specific sites of topoisomerase II activity that are initially detected in leptotene–zygotene spermatocytes just preceding the formation of the DNase I-sensitive domain. This has provided a likely model of the events initiating potentiation, i.e., the opening of a chromatin domain.

Nuclear organization of the protamine locus.

The human protamine gene cluster consists of three tightly regulated genes, protamine 1 (PRM1), protamine 2 (PRM2) and transition protein 2 (TNP2). Their products are required to repackage the paternal genome during spermiogenesis into a functional gamete. They reside within a single DNase I-sensitive domain associated with the sperm nuclear matrix, bounded by two haploid-specific Matrix Attachment Regions. The nuclear matrix is a dynamic proteinaceous network that is associated with both transcription and replication. While substantial effort has been directed toward pre- and post-transcriptional regulation, the role of the nuclear matrix in regulating haploid expressed genes has received comparatively little attention. In this regard, the functional organization of the human PRM1 --> PRM2 --> TNP2 cluster and where appropriate, comparisons to other model systems will be considered.

Tracking chromatin states using controlled DNase I treatment and real-time PCR

A novel approach to DNase I-sensitivity analysis was applied to examining genes of the spermatogenic pathway, reflective of the substantial morphological and genomic changes that occur during this program of differentiation. A new real-time PCR-based strategy that considers the nuances of response to nuclease treatment was used to assess the nuclease susceptibility through differentiation. Data analysis was automated with the K-Lab PCR algorithm, facilitating the rapid analysis of multiple samples while eliminating the subjectivity usually associated with Ct analyses. The utility of this assay and analytical paradigm as applied to nuclease-sensitivity mapping is presented.

Characterization of the Region Encompassing the Human Lysyl Oxidase Locus

A 46,823 bp region of human chromosome 5q23.1 encompassing the seven-exon lysyl oxidase gene was characterized at the primary sequence level. Approximately 17.4% of this region is comprised of repetitive elements. The gene colocalizes with microsatellite marker D5S467. It is flanked by two candidate nuclear matrix association regions (MARs). The 5′ MAR centered at position 12,500 is of the AT-rich and curved DNA class. This is followed by a large CpG island containing fifty-seven putative regulatory elements which extend from just upstream of exon 1 to intron 2. The larger 3′ MAR, spans position 35,050–39,750 and is characterized by a TG-rich kinked structure that also contains a topoisomerase II binding site. Based on these results model of the transcriptional regulation of the lysy/oxidase gene is presented.

Characterizing a human lysyl oxidase chromosomal domain

The expression of each locus in our genome is regulated by a gene-potentiative mechanism, whereby the gene first assumes the necessary structural conformation to enable transcription. This serves as the cornerstone for the three-tiered regulatory mechanism of potentiation, i.e., the opening of a chromatin domain, initiation of transcription, and transcript elongation. Although this is now generally accepted as the pathway that mediates gene expression, it has never been shown directly to control the expression of any heart-related gene. Lysyl oxidase enzymatically crosslinks members of the extracellular matrix, including elastin and collagen. Formation of these structures is essential to development and tissue repair. This system has enabled us to begin to address the underlying mechanism governing the selection of connective tissue genes for expression. However, before one can dissect this mechanism, it is necessary to define and characterize the locus, i.e., the corresponding genic domain. Our progress toward creating the resources necessary to unravel this mechanism is summarized in this review.