Phthalates impact on the epigenetic factors contributed specifically by the father at fertilization.

BACKGROUND: Preconception exposure to phthalates such as the anti-androgenic dibutyl-phthalate (DBP) impacts both male and female reproduction, yet how this occurs largely remains unknown. Previously we defined a series of RNAs expressly provided by sperm at fertilization and separately, and in parallel, those that responded to high DBP exposure. Utilizing both populations of RNAs, we now begin to unravel the impact of high-DBP exposure on those RNAs specifically delivered by the father. RESULTS: Enrichment of RNAs altered by DBP exposure within the Molecular Signature Database highlighted cellular stress, cell cycle, apoptosis, DNA damage response, and gene regulation pathways. Overlap within each of these five pathways identified those RNAs that were specifically (≥ fivefold enriched) or primarily (≥ twofold enriched) provided as part of the paternal contribution compared to the oocyte at fertilization. Key RNAs consistently altered by DBP, including CAMTA2 and PSME4, were delivered by sperm reflective of these pathways. The majority (64/103) of overlapping enriched gene sets were related to gene regulation. Many of these RNAs (45 RNAs) corresponded to key interconnected CRREWs (Chromatin remodeler cofactors, RNA interactors, Readers, Erasers, and Writers). Modeling suggests that CUL2, PHF10, and SMARCC1 may coordinate and mechanistically modulate the phthalate response. CONCLUSIONS: Mediated through a CRREW regulatory network, the cell responded to exposure presenting stressed-induced changes in the cell cycle-DNA damage-apoptosis. Interestingly, the majority of these DBP-responsive epigenetic mediators' direct acetylation or deacetylation, impacting the sperm's cargo delivered at fertilization and that of the embryo.

Cleavage of rRNA ensures translational cessation in sperm at fertilization.

Intact ribosomal RNAs (rRNAs) comprise the majority of somatic transcripts, yet appear conspicuously absent in spermatozoa, perhaps reflecting cytoplasmic expulsion during spermatogenesis. To discern their fate, total RNA retained in mature spermatozoa from three fertile donors was characterized by Next Generation Sequencing. In all samples, >75% of total sequence reads aligned to rRNAs. The distribution of reads along the length of these transcripts exhibited a high degree of non-uniformity that was reiterated between donors. The coverage of sequencing reads was inversely correlated with guanine-cytosine (GC)-richness such that sequences greater than ∼70% GC were virtually absent in all sperm RNA samples. To confirm the loss of sequence, the relative abundance of specific regions of the 28S transcripts in sperm was established by 7-Deaza-2'-deoxy-guanosine-5'-triphosphate RT-PCR. The inability to amplify specific regions of the 28S sequence from sperm despite the abundant representation of this transcript in the sequencing libraries demonstrates that approximately three-quarters of RNA retained in the mature male gamete are products of rRNA fragmentation. Hence, cleavage (not expulsion of the RNA component of the translational machinery) is responsible for preventing spurious translation following spermiogenesis. These results highlight the potential importance of those transcripts, including many mRNAs, which evade fragmentation and remain intact when sperm are delivered at fertilization. Sequencing data are deposited in GEO as: GSE29160.

Diverse system stresses: common mechanisms of chromosome fragmentation.

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Nuclear matrix association: switching to the invasive cytotrophoblast.

Abnormal trophoblast invasion is associated with the most common and most severe complications of human pregnancy. The biology of invasion, as well as the etiology of abnormal invasion remains poorly understood. The aim of this study was to characterize the transcriptome of the HTR-8/SVneo human cytotrophoblast cell line which displays well characterized invasive and non-invasive behavior, and to correlate the activity of the transcriptome with nuclear matrix attachment and cell phenotype. Comparison of the invasive to non-invasive HTR transcriptomes was unremarkable. In contrast, comparison of the MARs on chromosomes 14-18 revealed an increased number of MARs associated with the invasive phenotype. These attachment areas were more likely to be associated with silent rather than actively transcribed genes. This study supports the view that nuclear matrix attachment may play an important role in cytotrophoblast invasion by ensuring specific silencing that facilitates invasion.

Genomewide identification of nuclear matrix attachment regions: an analysis of methods.

High-throughput technologies now afford the opportunity to directly determine the distribution of MARs (matrix attachment regions) throughout a genome. The utility of cosmid and oligonucleotide platforms to identify human chromosome 16 MARs from preparations that employed LIS (lithium di-iodosalicylic acid) and NaCl extraction protocols was examined. The effectiveness of the platforms was then evaluated by Q-PCR (quantitative real-time PCR). Analysis revealed that caution must be exercised, since the representation of non-coding regions varies among platforms. Nevertheless, several interesting trends were revealed. We expect that these technologies will prove useful in systems approaches directed towards defining the role of MARs in various cell types and cellular processes.

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.

LARALink: a web application for cytogenetic linkage analysis.

Genomic and expression data have increased dramatically over the last several years. This is primarily due to the completion of the human genome project as well as an upsurge in the use of various high-throughput technologies. Recent attempts to correlate genomic and expression data have stimulated the scientific community to determine how this data can be used within a clinical setting (P Khatri et al., Genomics 2002: 79: 266; LJ van't Veer et al., Nature 2002: 415: 530). LARALink (Loci Analysis for Rearrangements Link) is a database-driven web application that utilizes several public datasets to analyze clinical cytogenetic data to identify candidate genes. LARALink allows UniGene clusters or single-nucleotide polymorphisms (SNPs) to be queried for multiple patients by cytoband, chromosome marker, or base pair. The results can be further refined with the use of an anatomical site, developmental stage, pathology, or cell-type expression filter. Once a set of UniGene clusters (expressed genes) has been identified either for a single patient or for a shared region among multiple patients, the expression-distribution profile, expressed sequence tags (ESTs), or online mendelian inheritance in man (OMIM) entries are displayed. The utility of this tool is shown by its application to both research and clinical medicine. LARALink is a public resource available at: http://www.laralink.bioinformatics.wayne.edu:8080/unigene.

From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics.

Eukaryotic genomes are functionally compartmentalized into chromatin domains by their attachment to a supporting structure that has traditionally been termed the nuclear matrix. Present evidence indicates the dynamics of this entity, which requires particular properties of the elements that mediate this kind of interaction. Above all, this is enabled by the so-called 'mass binding phenomenon' by which scaffold/matrix-attachment regions (S/MARs) reversibly associate with ubiquitous factors. Recent investigations and novel techniques have shown that these contacts can be altered by modulators as well as by specific interactions with the components of enhancers and locus control regions.

Analysis of marker or complex chromosomal rearrangements present in pre- and post-natal karyotypes utilizing a combination of G-banding, spectral karyotyping and fluorescence in situ hybridization.

The significance of complex chromosomal rearrangements presents a diagnostic dilemma. In the past, the use of G-banding coupled with fluorescence in situ hybridization (FISH) has been the standard approach. The recent development of spectral karyotyping (SKY) and multicolor FISH (M-FISH) has resulted in an increased accuracy of identification of marker or other complex chromosomal rearrangements. However, owing to the additional cost and time associated with SKY or M-FISH, and the restricted availability of such imaging facilities in many centers, it is not feasible to perform these procedures routinely on every sample. In addition, the identification of an aberration by SKY or M-FISH will often require confirmation by FISH. A practical approach is needed to take advantage of the complementary strengths of each method. In our center we utilize an algorithm that dictates the use of routine G-banding for the initial preliminary evaluation of a patient, followed by SKY characterization if marker chromosomes or complex translocations are detected by the G-banding analysis. According to this algorithm, FISH is used to verify the results once the origin of the abnormal chromosome has been determined by SKY. To demonstrate the effectiveness of this algorithm, we have analyzed both amniocyte and lymphocyte slides, using a combination of G-banding, SKY, and FISH. Our results confirm that an algorithm which selectively uses SKY or M-FISH will provide an efficient and improved method for pre- and post-natal chromosomal analysis.

Type II gonadotropin-releasing hormone receptor transcripts in human sperm.

GnRH regulates reproduction via the well-characterized mammalian pituitary GnRH receptor (type I). In addition, two homologous genes for a second form of the GnRH receptor (type II) are present in the human genome, one on chromosome 14 and the second on chromosome 1. The chromosome 14 gene is ubiquitously transcribed at high levels in the antisense orientation but lacks exon 1, required to encode a full-length receptor. In comparison, the chromosome 1 gene contains all three exons. The issue of whether this gene is transcribed in any human tissue(s), and whether these transcripts encode a functional receptor protein, remains unresolved. We have directly addressed this by screening a panel of human RNAs by hybridization and RT-PCR. These analyses showed that, unlike the chromosome 14 gene, chromosome 1 gene expression is limited and of low abundance. Exon 1-containing transcripts were detected by in situ hybridization in mature sperm and in human postmeiotic testicular cells. Further sequence analysis revealed that although all the potential coding segments were present, the human transcripts, like the gene, contain a stop codon within the coding region and a frame-shift relative to other mammalian GnRH receptors. Although this suggests that the human gene may be a transcribed pseudogene, a functional type II GnRH receptor cDNA has recently been cloned from monkeys. Given the well-established role of GnRH in spermatogenesis and reported evidence of type II GnRH receptor immunoreactivity in human tissues, it is possible that the chromosome 1 gene is functional.

Sperm nuclear matrix association of the PRM1-->PRM2-->TNP2 domain is independent of Alu methylation.

Genes or multigenic chromosomal regions are organized by the nuclear matrix into a series of functionally discrete genic domains. Biophysical analysis of the human chromosome 16p13.13 region has shown that the PRM1-->PRM2-->TNP2 protamine containing multigenic locus is bounded by two sperm nuclear matrix attachment regions (MAR). This domain exists in a transcriptionally readied or potentiated (i.e. open) chromatin state when associated with the nuclear matrix. The MAR-bounded PRM1-->PRM2-->TNP2 locus is nestled in an Alu repetitive element dense region. Fluorescence in-situ hybridization, analysis of sperm nuclear matrix/halo preparations showed that the PRM1-->PRM2-->TNP2 domain specifically localizes to the sperm nuclear matrix. This raised the question of whether nuclear matrix association and gene expression in this locus is mediated by Alu methylation. The methylation status of the various Alu elements contained within the human PRM1-->PRM2-->TNP2 locus was therefore assayed. The seven Alu elements tested, including those associated with the matrix attachment regions within the PRM1-->PRM2-->TNP2 locus, were fully methylated in sperm DNA. Conversely, these same Alu repeats were hypomethylated within the erythroleukaemic cell line, K562, which does not express any of the genes from this domain. This study shows that Alu methylation status is independent of attachment of PRM1-->PRM2-->TNP2 locus to the nuclear matrix and that Alu methylation does not play a leading role in the regulation of this domain.

Re-defining the chromatin loop domain.

It is commonly accepted that the loop domain represents the basic structural unit of eukaryotic chromatin associated with DNA replication, gene expression and higher order packaging. However, molecular-cytological information defining the loop domain is lacking. There are gaps in our knowledge of the loop structure and how it regulates gene expression. The combination of new data/reagents from the Human Genome Project plus the use of novel molecular cytological technology will provide answers. Here we briefly review the status of chromatin loop research and pose questions that need to be addressed. New experimental systems are also presented to target some long-standing issues regarding the structure and function of the chromatin loop domain and its relationship with the nuclear matrix. This new knowledge will have a profound impact for modern genetics and molecular medicine.

The combination of SKY and specific loci detection with FISH or immunostaining.

Spectral karyotyping (SKY) represents an effective tool to detect individual chromosomes and analyze major karyotype abnormalities within an entire genome. We have tested the feasibility of combining SKY and FISH/protein detection in order to combine SKY's unique abilities with specific loci detection. Our experimental results demonstrate that various combined protocols involving SKY, FISH and immunostaining work well when proper procedures are used. This combined approach allows the tracking of key genes or targeted chromosome regions while monitoring changes throughout the whole genome. It is particularly useful when simultaneously monitoring the behavior of both protein complexes and DNA loci within the genome. The details of this methodology are described and systematically tested in this communication.

Recovering filter-based microarray data for pathways analysis using a multipoint alignment strategy.

The use of commercial microarrays is rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of biological and software tools to the research community for these analyses. The fidelity of data analysis using these tools is dependent on a series of well-defined reference control points in the array. During the course of our investigations, it became apparent that in some instances the reference control points that are required for analysis became lost in background noise. This effectively halted the analysis and the recovery of any information contained within that experiment. To recover this data and to increase analytical veracity, the simple strategy of superimposing a template of reference control points onto the experimental array was developed. The utility of this tool is established in this communication.

Design and implementation of an introductory course for computer applications in molecular genetics. A case study.

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campus-wide computational biology course to a new and widely diverse student body.

Development of a 950-gene DNA array for examining gene expression patterns in mouse testis.

BACKGROUND: Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own. RESULTS: We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes. CONCLUSION: We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.

Whole-body gene expression by data mining.

To date, a comprehensive survey of the expression of lysyl oxidase (LOX), lysyl oxidase-like 1 (LOXL1), and lysyl oxidase-like 2 (LOXL2) has yet to be performed. The use of in vitro strategies to accomplish this task would prove daunting as it is both time-consuming and costly. We present a new in silico data mining strategy that directly addresses these limitations. Sequences corresponding to the 3' untranslated regions of LOX, LOXL1, and LOXL2 were individually queried against the human expressed sequence tag database (dbEST). In this manner, the entire tissue repertoire available in the dbEST was surveyed. This provided an estimate of the levels of mRNA transcripts in a variety of adult and fetal tissues. We have also employed this strategy to determine the pattern of expression and levels of a newly discovered gene, CGI-15. The veracity of this technique has been independently assessed by semiquantitative PCR analysis. The application of this technology is bounded only by the ever-growing information available in the GenBank, UniGene, and human EST databases. The utility of our data mining strategy to establish relative transcript levels in numerous tissues 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 corner-stone 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.