Anatomy and evolution of a DNA replication origin.

DNA amplification occurs at the DNA puff II/9A locus in the fungus fly Sciara coprophila. As a foundation to study the molecular mechanism for the initiating events of II/9A DNA re-replication, we have sequenced 14 kb spanning a DNase hypersensitive site (DHS) upstream of the 1 kb amplification origin and through transcription units II/9-1 and II/9-2 downstream of the origin. These elements are annotated as well as the ORC binding site at the origin and the transition point (TP) between continuous and discontinuous DNA syntheses that marks the origin of bidirectional replication at the nucleotide level. A 9 bp motif found at the TP is repeated near the other end of the 1 kb ORI and may identify a putative second TP. The steroid hormone ecdysone induces DNA amplification as well as transcription and puffing at locus II/9A. Within the 14 kb, several matches to the ecdysone response element (EcRE) consensus sequence were identified, including some in the amplification origin region. EcRE O-P is at a central axis of a remarkable symmetry, equidistant to the TPs that are themselves equidistant to EcRE O-1 and EcRE O-2. DNA sequence alterations have occurred throughout the II/9A region in a newly discovered polymorphism (#2). Polymorphism #2 is not specific to developmental stage, sex, or tissue, and it does not impair DNA amplification. The DHS, both 9 bp TP sequences, and EcREs O-1, O-P, and O-2 are conserved between the polymorphism #1 and #2 sequences, suggesting their functional importance and retention during evolutionary selection. Moreover, a 72 bp sequence in the Sciara DHS at DNA puff II/9A is conserved in DNA puff C-3 of Rhynchosciara americana. Comparisons are discussed between the Sciara II/9A amplicon and the chorion locus amplicon on the third chromosome of Drosophila.

ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.

TAF4b Regulates Oocyte-Specific Genes Essential for Meiosis.

TAF4b is a gonadal-enriched subunit of the general transcription factor TFIID that is implicated in promoting healthy ovarian aging and female fertility in mice and humans. To further explore the potential mechanism of TAF4b in promoting ovarian follicle development, we analyzed global gene expression at multiple time points in the human fetal ovary. This computational analysis revealed coordinate expression of human TAF4B and critical regulators and effectors of meiosis I including SYCP3, YBX2, STAG3, and DAZL. To address the functional relevance of this analysis, we turned to the embryonic Taf4b-deficient mouse ovary where, for the first time, we demonstrate, severe deficits in prophase I progression as well as asynapsis in Taf4b-deficient oocytes. Accordingly, TAF4b occupies the proximal promoters of many essential meiosis and oogenesis regulators, including Stra8, Dazl, Figla, and Nobox, and is required for their proper expression. These data reveal a novel TAF4b function in regulating a meiotic gene expression program in early mouse oogenesis, and support the existence of a highly conserved TAF4b-dependent gene regulatory network promoting early oocyte development in both mice and women.

TAF4b is required for mouse spermatogonial stem cell development.

Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here, we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild-type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development.

Piwi regulates Vasa accumulation during embryogenesis in the sea urchin.

BACKGROUND: Piwi proteins are essential for germ line development, stem cell maintenance, and more recently found to function in epigenetic and somatic gene regulation. In the sea urchin Strongylocentrotus purpuratus, two Piwi proteins, Seawi and Piwi-like1, have been identified, yet their functional contributions have not been reported. RESULTS: Here we found that Seawi protein was localized uniformly in the early embryo and then became enriched in the primordial germ cells (PGCs) (the small micromere lineage) from blastula stage and thereafter. Morpholino knockdown of Sp-seawi diminished PGC-specific localization of Seawi proteins, and altered expression of other germ line markers such as Vasa and Gustavus, but had no effect on Nanos. Furthermore, Seawi knockdown transiently resulted in Vasa positive cell proliferation in the right coelomic pouch that appear to be derived from the small micromere lineage, yet they quickly disappeared with an indication of apoptosis by larval stage. Severe Seawi knockdown resulted in an increased number of apoptotic cells in the entire gut area. CONCLUSION: Piwi proteins appear to regulate PGC proliferation perhaps through control of Vasa accumulation. In this organism, Piwi is likely regulating mRNAs, not just transposons, and is potentially functioning both inside and outside of the germ line during embryogenesis.

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ-line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.

Meiotic gene expression initiates during larval development in the sea urchin.

BACKGROUND: Meiosis is a unique mechanism in gamete production and a fundamental process shared by all sexually reproducing eukaryotes. Meiosis requires several specialized and highly conserved genes whose expression can also identify the germ cells undergoing gametogenic differentiation. Sea urchins are echinoderms, which form a phylogenetic sister group of chordates. Sea urchin embryos undergo a feeding, planktonic larval phase in which they construct an adult rudiment prior to metamorphosis. Although a series of conserved meiosis genes (e.g., dmc1, msh5, rad21, rad51, and sycp1) is expressed in sea urchin oocytes, we sought to determine when in development meiosis would first be initiated. RESULTS: We surveyed the expression of several meiotic genes and their corresponding proteins in the sea urchin Strongylocentrotus purpuratus. Surprisingly, meiotic genes are highly expressed not only in ovaries but beginning in larvae. Both RNA and protein localizations strongly suggest that meiotic gene expression initiates in tissues that will eventually give rise to the adult rudiment of the late larva. CONCLUSIONS: These results demonstrate that broad expression of the molecules associated with meiotic differentiation initiates prior to metamorphosis and may have additional functions in these cells, or mechanisms repressing their function, until later in development when gametogenesis begins.

Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis.

Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development.

Immunogenic Consensus Sequence T helper Epitopes for a Pan-Burkholderia Biodefense Vaccine.

BACKGROUND: Biodefense vaccines against Category B bioterror agents Burkholderia pseudomallei (BPM) and Burkholderia mallei (BM) are needed, as they are both easily accessible to terrorists and have strong weaponization potential. Burkholderia cepaciae (BC), a related pathogen, causes chronic lung infections in cystic fibrosis patients. Since BPM, BM and BC are all intracellular bacteria, they are excellent targets for T cell-based vaccines. However, the sheer volume of available genomic data requires the aid of immunoinformatics for vaccine design. Using EpiMatrix, ClustiMer and EpiAssembler, a set of immunoinformatic vaccine design tools, we screened the 31 available Burkholderia genomes and performed initial tests of our selections that are candidates for an epitope-based multi-pathogen vaccine against Burkholderia species. RESULTS: Immunoinformatics analysis of 31 Burkholderia genomes yielded 350,004 9-mer candidate vaccine peptides of which 133,469 had perfect conservation across the 10 BM genomes, 175,722 had perfect conservation across the 11 BPM genomes and 40,813 had perfect conservation across the 10 BC genomes. Further screening with EpiMatrix yielded 54,010 high-scoring Class II epitopes; these were assembled into 2,880 longer highly conserved 'immunogenic consensus sequence' T helper epitopes. 100% of the peptides bound to at least one HLA class II allele in vitro, 92.7% bound to at least two alleles, 82.9% to three, and 75.6% of the binding results were consistent with the immunoinformatics analysis. CONCLUSIONS: Our results show it is possible to rapidly identify promiscuous T helper epitopes conserved across multiple Burkholderia species and test their binding to HLA ligands in vitro. The next step in our process will be to test the epitopes ex vivo using peripheral leukocytes from BC, BPM infected humans and for immunogenicity in human HLA transgenic mice. We expect that this approach will lead to development of a licensable, pan-Burkholderia biodefense vaccine.

Vasa genes: emerging roles in the germ line and in multipotent cells.

Sexually reproducing metazoans establish a cell lineage during development that is ultimately dedicated to gamete production. Work in a variety of animals suggests that a group of conserved molecular determinants act in this germ line maintenance and function. The most universal of these genes are Vasa and Vasa-like DEAD-box RNA helicase genes. However, recent evidence indicates that Vasa genes also function in other cell types, distinct from the germ line. Here we evaluate our current understanding of Vasa function and its regulation during development, addressing Vasa's emerging role in multipotent cells. We also explore the evolutionary diversification of the N-terminal domain of this gene and how this impacts the association of Vasa with nuage-like perinuclear structures.

Polycomb group gene expression in the sea urchin.

Embryonic development in metazoan animals involves a remarkable degree of both genetic and epigenetic regulation. Polycomb group (PcG) genes are essential for initiation and maintenance of epigenetic developmental regulation. This report identifies homologous PcG genes representing the core components of three known Polycomb repressive complexes in the sea urchin Strongylocentrotus purpuratus. Quantitative, temporal, and spatial analyses revealed two significant aspects of their transcript expression during embryonic development. First, almost all showed a localized expression pattern in mesenchyme blastula and gastrula-stage embryos. As the embryo specifies its three germ layers, it may require its PcG gene transcripts to remain in localized stem cell populations, but turn-over in differentiating tissues. Second, their transcript levels gradually decreased during larval pluteus stages. This finding is consistent with progressive cell differentiation in the embryo. Taken together, these results suggest that PcG genes are conserved in S. purpuratus and are actively expressed during early embryogenesis.