Rongjia Zhou

Multiple Alternative Splicing and Differential Expression of dmrt1 During Gonad Transformation of the Rice Field Eel

Abstract Morphologically distinct males and females are observed throughout the animal kingdom. Why and how sex evolved and is maintained in most living organisms remains a key question in cellular and evolutionary biology. Here we report that four isoforms of dmrt1 (dsx- and mab3-related transcription factor 1) are generated in testis, ovotestis, and ovary by alternative splicing in the rice field eel, a fresh water fish that undergoes natural sex reversal from female to male during its life cycle. These transcripts encode four different size proteins with 301, 196, 300, and 205 amino acids. Like fly doublesex splicing, the dmrt1 of the rice field eel is also alternatively spliced at the 3′ region, which generates diverse isoforms in gonads by alternative use of 3′ sequences. Not only is dmrt1 expressed specifically in gonads, but its multiple isoforms are differentially coexpressed in gonadal epithelium during gonad transformation. Expression levels of a and b isoforms of dmrt1 ranged from low to high (ovary < ovotestis I < ovotestis II < ovotestis III < testis), based on comparisons of mean values from real-time fluorescent quantitative reverse transcription-polymerase chain reaction analysis. The overall expression level of dmrt1 b was much lower than that of dmrt1 a. Expression of dmrt1 d was not only low, but it also did not change significantly during sex transformation. The differential expression of dmrt1 isoforms may also be regulated by their 3′ untranslated regions (UTRs), although these 3′ UTRs do not contribute to intracellular localization of the Dmrt1 protein. These results provide new insight into roles of regulation at the level of splicing of dmrt1 in governing the sex differentiation cascade.

A global view of cancer-specific transcript variants by subtractive transcriptome-wide analysis.

Background Alternative pre-mRNA splicing (AS) plays a central role in generating complex proteomes and influences development and disease. However, the regulation and etiology of AS in human tumorigenesis is not well understood. Methodology/Principal Findings A Basic Local Alignment Search Tool database was constructed for the expressed sequence tags (ESTs) from all available databases of human cancer and normal tissues. An insertion or deletion in the alignment of EST/EST was used to identify alternatively spliced transcripts. Alignment of the ESTs with the genomic sequence was further used to confirm AS. Alternatively spliced transcripts in each tissue were then subtractively cross-screened to obtain tissue-specific variants. We systematically identified and characterized cancer/tissue-specific and alternatively spliced variants in the human genome based on a global view. We identified 15,093 cancer-specific variants of 9,989 genes from 27 types of human cancers and 14,376 normal tissue-specific variants of 7,240 genes from 35 normal tissues, which cover the main types of human tumors and normal tissues. Approximately 70% of these transcripts are novel. These data were integrated into a database HCSAS (http://202.114.72.39/database/human.html, pass:68756253). Moreover, we observed that the cancer-specific AS of both oncogenes and tumor suppressor genes are associated with specific cancer types. Cancer shows a preference in the selection of alternative splice-sites and utilization of alternative splicing types. Conclusions/Significance These features of human cancer, together with the discovery of huge numbers of novel splice forms for cancer-associated genes, suggest an important and global role of cancer-specific AS during human tumorigenesis. We advise the use of cancer-specific alternative splicing as a potential source of new diagnostic, prognostic, predictive, and therapeutic tools for human cancer. The global view of cancer-specific AS is not only useful for exploring the complexity of the cancer transcriptome but also widens the eyeshot of clinical research.

Differential genome duplication and fish diversity

The duplication of genes and entire genomes arebelieved to be important mechanisms underlyingmorphological variation and functionalinnovation in the evolution of life andespecially for the broad diversity observed inthe speciation of fishes. How did these fishspecies and their genetic diversity arise? Theoccurrence of three rounds of genomeduplication during vertebrate evolution mightexplain why many gene families are typicallyabout half the size in land vertebrates as theyare in fishes. However, mechanisms of geneticdiversity in fish lineages need to be furtherexplained. Here we propose that differentialgenome duplication of from two to six roundsoccurred in different fish lines, offering newopportunities during the radiation of fishlineages. This model provides a fundamentalbasis for the understanding of theirspeciation, diversity and evolution.

SRY-related genes in the genome of the rice field eel (Monopterus albus).

The mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMG-box gene) genes. Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice field eel, Monopterus albus, and DNA sequence information of the HMG box region of five Sox genes. The Sox 1, Sox 4 and Sox 14 genes do not have introns in the HMG box region. The Sox 9 gene and Sox 17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox 9 and Sox 17 genes among mammals, chickens and fish suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation.

Molecular cloning and expression of Sox17 in gonads during sex reversal in the rice field eel, a teleost fish with a characteristic of natural sex transformation.

The Sox is a large family of genes which encode transcription factors with high-mobility-group DNA binding domain related to the SRY, with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the identification of Sox17 gene of the rice field eel, a teleost fish with a characteristic of natural sex transformation. This gene, located on chromosome 5, consists of two exons which encode a 399-amino acid protein with a conserved HMG box. Phylogenetic analysis shows that the rice field eel Sox17 fits within the Sox17 clade of vertebrates. The rice field eel Sox17 was dominantly expressed in gonads of male, female, and intersex, besides in brain and spleen. Gene expression analysis by in situ hybridization showed its expression in testis, ovary, and ovotestis, and specifically in the gonadal lamellae of ovary, ovotestis, and testis and developing spermatogenic cells of testis, suggesting that they have potentially important roles in gonadal differentiation during sex reversal in this species.

DNA demethylation and USF regulate the meiosis-specific expression of the mouse Miwi.

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type–specific expression of Miwi. CpG methylation at the USF–binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter–driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis.

Multiple alternative splicing in gonads of chicken DMRT1.

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla, although the existence of two sexes is nearly universal in the animal kingdom. However, the evolutionarily conserved DMRT1/dsx/mab3 with a common zinc finger-like DNA-binding motif, DM domain, share both similar structure and function between phyla. Here we report that six transcripts of the chicken DMRT1 were generated in gonads by multiple alternative splicing. By cDNA cloning and genomic structure analysis, we found that there were nine exons of DMRT1, which were involved in alternatively splicing to generate the DMRT1 transcripts. Northern blotting and reverse transcription (RT) PCR analysis revealed that the expression of chicken DMRT1 was testis-specific in adults. Whole-mount in situ hybridizations and RT-PCR indicated that DMRT1 b was specially expressed in embryo gonads and higher in male than female gonads at stage 31. The female gonad had stronger DMRT1 c expression than the male one, whereas DMRT1 f was detectable only in the male gonad at stage 31 of the key time of sex gonadal differentiation. The differential expression of these transcripts during gonadal differentiation provides new insight into roles of alternative splicing of DMRT1 in governing sex differentiation of the chicken.

The rice field eel as a model system for vertebrate sexual development

Complex developmental mechanisms of vertebrates are unraveled using comparative genomic approaches. Several teleosts, such as zebrafish, medaka and pufferfish, are used as genetic model systems because they are amenable to studies of gene function. The rice field eel, a freshwater fish, is emerging as a specific model system for studies of vertebrate sexual development because of its small genome size and naturally occurring sex reversal. Data presented here support the use of the rice field eel as another important fish model for comparative genome studies, especially in vertebrate sexual development. This model system is complementary rather than redundant.

Transcriptional diversity of DMRT1 (dsx- and mab3-related transcription factor 1) in human testis

Recent advances in the evolutionary genetics of sex determination indicate that the only molecular similarity in sex determination found so far among phyla is between the fly doublesex, worm mab-3 and vertebrate DMRT1(dsx- and mab3-related transcription factor 1) /DMY genes. Each of these factors encodes a zinc-finger-like DNA-binding motif, DM domain. Insights into the evolution and functions of human DMRT1 gene could reveal evolutionary mechanisms of sexual development. Here we report the identification and characterization of multiple isoforms of human DMRT1 in the testis. These transcripts encode predicted proteins with 373, 275 and 175 amino acids and they were generated by alternative splicing at 3′ region. Expression level of DMRT1a is higher than those of both DMRT1b and c, and the DMRT1c expression was the lowest in testis, based on comparisons of mean values from real-time fluorescent quantitative RT-PCR analysis. Both DMRT1b and c result from exonization of intronic sequences, including the exonization of an Alu element. A further search for Alu elements within the DMRT1 gene demonstrated that all 99 Alu elements are non-randomly distributed among the non-coding regions on both directions. These new characteristics of DMRT1 would have an important impact on the evolution of sexual development mechanisms.

Nuclear localization, DNA binding and restricted expression in neural and germ cells of zebrafish Dmrt3

Background information. The DM (doublesex and male aberrant‐3) genes implicated in sexual development in diverse metazoan organisms have been proved to be involved in development of non‐gonadal tissues. The aim of the present study was to identify and characterize Dmrt3 (DM‐related transcription factor 3) of zebrafish.