Assembly of Drosophila Centromeric Chromatin Proteins during Mitosis

Semi-conservative segregation of nucleosomes to sister chromatids during DNA replication creates gaps that must be filled by new nucleosome assembly. We analyzed the cell-cycle timing of centromeric chromatin assembly in Drosophila, which contains the H3 variant CID (CENP-A in humans), as well as CENP-C and CAL1, which are required for CID localization. Pulse-chase experiments show that CID and CENP-C levels decrease by 50% at each cell division, as predicted for semi-conservative segregation and inheritance, whereas CAL1 displays higher turnover. Quench-chase-pulse experiments demonstrate that there is a significant lag between replication and replenishment of centromeric chromatin. Surprisingly, new CID is recruited to centromeres in metaphase, by a mechanism that does not require an intact mitotic spindle, but does require proteasome activity. Interestingly, new CAL1 is recruited to centromeres before CID in prophase. Furthermore, CAL1, but not CENP-C, is found in complex with pre-nucleosomal CID. Finally, CENP-C displays yet a different pattern of incorporation, during both interphase and mitosis. The unusual timing of CID recruitment and unique dynamics of CAL1 identify a distinct centromere assembly pathway in Drosophila and suggest that CAL1 is a key regulator of centromere propagation

TAF4b promotes mouse primordial follicle assembly and oocyte survival

AbstractPrimary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development

Dynamic transcriptome profiles within spermatogonial and spermatocyte populations during postnatal testis maturation revealed by single-cell sequencing.

Spermatogenesis is the process by which male gametes are formed from a self-renewing population of spermatogonial stem cells (SSCs) residing in the testis. SSCs represent less than 1% of the total testicular cell population in adults, but must achieve a stable balance between self-renewal and differentiation. Once differentiation has occurred, the newly formed and highly proliferative spermatogonia must then enter the meiotic program in which DNA content is doubled, then halved twice to create haploid gametes. While much is known about the critical cellular processes that take place during the specialized cell division that is meiosis, much less is known about how the spermatocytes in the "first-wave" in juveniles compare to those that contribute to long-term, "steady-state" spermatogenesis in adults. Given the strictly-defined developmental process of spermatogenesis, this study explored the transcriptional profiles of developmental cell stages during testis maturation. Using a combination of comprehensive germ cell sampling with high-resolution, single-cell-mRNA-sequencing, we have generated a reference dataset of germ cell gene expression. We show that discrete developmental stages of spermatogenesis possess significant differences in the transcriptional profiles from neonates compared to juveniles and adults. Importantly, these gene expression dynamics are also reflected at the protein level in their respective cell types. We also show differential utilization of many biological pathways with age in both spermatogonia and spermatocytes, demonstrating significantly different underlying gene regulatory programs in these cell types over the course of testis development and spermatogenic waves. This dataset represents the first unbiased sampling of spermatogonia and spermatocytes during testis maturation, at high-resolution, single-cell depth. Not only does this analysis reveal previously unknown transcriptional dynamics of a highly transitional cell population, it has also begun to reveal critical differences in biological pathway utilization in developing spermatogonia and spermatocytes, including response to DNA damage and double-strand breaks

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 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

<i>Taf4b</i>-deficient oocytes experience defective meiotic progression and chromosome asynapsis.

<p>Pachytene spreads were prepared using the drying-down technique (50) on cell suspensions from E16.5 wild-type (A-D; I-L) and <i>Taf4b</i>-deficient (E-H; M-P) ovaries. Slides were stained for SYCP1 and SYCP3 (A-H), or SYCP3 and CENP-A (I-P). White arrowheads in (H) indicate regions of asynapsis in which SYCP3 is localized but SYCP1 is not; while white arrowheads in (P) indicate regions of asynapsis, many of which can be visualized by non-overlapping CENP-A centromeric foci. Slides were also stained for γH2AX and SYCP3 (Q, R) to visualize double-strand breaks. A high incidence of asynapsis (S), as scored by >20 CENP-A foci, as well as defects in meiotic progression (T), as scored by SYCP3 configuration (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006128#pgen.1006128.s001" target="_blank">S1 Fig</a>), were apparent in <i>Taf4b</i>-deficient oocytes. *: n = 4 animals each, one-tailed t-test, p<0.05</p

Reduced MSY2 expression and diplotene arrest in <i>Taf4b</i>-deficient oocytes.

<p>(A) PND0 wild-type and <i>Taf4b</i>-deficient ovary tissue sections were stained with primary antibodies against MSY2 and germ cell marker TRA98, which were then quantified for the number of MSY2+/TRA98+ double-positive cells (B). As MSY2 indicates diplotene arrest, the data suggest a defect in diplotene arrival in <i>Taf4b</i>-deficient oocytes.</p

Model of TAF4b promoting a critical meiosis and oogenesis gene regulatory network: Data from chromatin immunoprecipitation experiments demonstrates that TAF4b occupies the proximal promoters of <i>Dazl</i>, <i>Stra8</i>, <i>Figlα</i>, and <i>Nobox</i>, ultimately resulting in their expression.

<p>Proper expression of these essential regulators facilitates expression of downstream meiosis and oogenesis genes, finally leading to the development of a healthy primordial follicle pool. Red arrows represent direct transcriptional regulation, gray arrows represent post-transcriptional regulation, and dashed lines indicate putative mechanisms.</p