Kimberly A. Seymour

The chromatin-targeting protein Brd2 is required for neural tube closure and embryogenesis.

Chromatin modifications are essential for directing transcription during embryonic development. Bromodomain-containing protein 2 (Brd2; also called RING3 and Fsrg1) is one of four BET (bromodomain and extra-terminal domain) family members known to selectively bind acetylated histones H3 and H4. Brd2 associates with multiple subunits of the transcriptional apparatus including the mediator, TFIID and Swi/Snf multiprotein complexes. While molecular interactions of Brd2 are known, the functions of Brd2 in mammalian embryogenesis remain unknown. In developing a mouse model deficient in Brd2, we find that Brd2 is required for the completion of embryogenesis and proper neural tube closure during development. Embryos lacking Brd2 expression survive up to embryonic day 13.5, soon after mid-gestation, and display fully penetrant neurulation defects that largely result in exencephaly of the developing hindbrain. In this study, we find that highest expression of Brd2 is detected in the developing neural tube, correlating with the neural tube defects found in Brd2-null embryos. Additionally, embryos lacking Brd2 expression display altered gene expression programs, including the mis-expression of multiple genes known to guide neuronal development. Together these results implicate essential roles for Brd2 as a critical integrator of chromatin structure and transcription during mammalian embryogenesis and neurogenesis.

Accelerated Ovarian Aging in the Absence of the Transcription Regulator TAF4B in Mice

Abstract The mammalian ovary is unique in that its reproductive life span is limited by oocyte quantity and quality. Oocytes are recruited from a finite pool of primordial follicles that are usually exhausted from the ovary during midadult life. If regulation of this pool is perturbed, the reproductive capacity of the ovary is compromised. TAF4B is a gonad-enriched subunit of the TFIID complex required for female fertility in mice. Previous characterization of TAF4B-deficient ovaries revealed several reproductive deficits that collectively result in infertility. However, the etiology of such fertility defects remains unknown. By assaying estrous cycle, ovarian pathology, and gene expression changes in young Taf4b-null female mice, we show that TAF4B-deficient female mice exhibit premature reproductive senescence. The rapid decline of ovarian function in Taf4b-null mice begins in early postnatal life, and follicle depletion is completed by 16 wk of age. To uncover differences in gene expression that may underlie accelerated ovarian aging, we compared genome-wide expression profiles of 3-wk-old, prepubescent Taf4b-null and wild-type ovaries. At 3 wk of age, decreased gene expression in Taf4b-null ovaries is similar to that seen in aged ovaries, revealing several molecular signatures of premature reproductive senescence, including reduced Smc1b. One significantly reduced transcript in the young TAF4B-null ovary codes for MOV10L1, a putative germline-specific RNA helicase that is related to the Drosophila RNA interference protein, armitage. We show here that Mov10l1 is expressed in mouse oocytes and that its expression is sensitive to TAF4B level, linking TAF4B to the posttranscriptional control of ovarian gene expression.

TAF4b promotes mouse primordial follicle assembly and oocyte survival.

Primary 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.

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. Stem Cells 2015;33:1267–1276

Estrogen Responsiveness of the TFIID Subunit TAF4B in the Normal Mouse Ovary and in Ovarian Tumors

ABSTRACT Estrogen signaling in the ovary is a fundamental component of normal ovarian function, and evidence also indicates that excessive estrogen is a risk factor for ovarian cancer. We have previously demonstrated that the gonadally enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice and for the proliferation of ovarian granulosa cells following hormonal stimulation. However, the relationship between TAF4B and estrogen signaling in the normal ovary or during ovarian tumor initiation and progression has yet to be defined. Herein, we show that Taf4b mRNA and TAF4B protein, but not Taf4a mRNA or TAF4A protein, are increased in whole ovaries and granulosa cells of the ovary after exposure to 17beta-estradiol or the synthetic estrogen diethylstilbestrol and that this response occurs within hours after stimulation. Furthermore, this increase occurs via nuclear estrogen receptors both in vivo and in a mouse granulosa cancer cell line, NT-1. We observe a significant increase in Taf4b mRNA in estrogen-supplemented mouse ovarian tumors, which correlates with diminished survival of these mice. These data highlight the novel response of the general transcription factor TAF4B to estrogen in the normal ovary and during ovarian tumor progression in the mouse, suggesting its potential role in regulating actions downstream of estrogen stimulation.

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.

Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

Dopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11−27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required. Note All research was planned and conducted while members were at Brown University Research funding NIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

Abstract A81: Estrogen-responsiveness of the TFIID subunit TAF4B and its potential function in ovarian cancer, epigenetic regulation and meiotic DNA repair

Female infertility affects approximately 10.9% of women ages 15-44 in the US, and the molecular mechanisms leading to this disorder are multifaceted and varied. We have previously demonstrated that the gonadal-enriched TFIID subunit TAF4B, a paralog of the general transcription factor TAF4A, is required for fertility in mice. Female mice deficient for TAF4B exhibit a phenotype resembling premature ovarian failure, including early oocyte loss, follicular atresia and severely reduced granulosa cell proliferation when treated with 17β-estradiol. The inability of estrogen to stimulate granulosa cell proliferation led us to hypothesize that TAF4B is involved in an estrogen signaling pathway within the ovary. A large percentage of Taf4b -knockout oocytes die by apoptosis immediately after birth, and this germ cell loss is attenuated by estrogen supplementation, further suggesting a connection between TAF4B and estrogen. Taf4b -knockout ovaries also display deregulated epigenetic marks, which can affect DNA repair during meiotic homologous recombination. Furthermore, estrogen is known to regulate epigenetic changes in the ovary, leading us to hypothesize that the estrogen rescue may occur via modulation of the epigenetic state and consequent repair of double-strand breaks during meiosis. Here, we show that Taf4b mRNA and TAF4B protein expression are upregulated by estrogens in whole ovaries and purified granulosa cells of the ovary and that this increase occurs via nuclear estrogen receptors. We observe significant increases of Taf4b mRNA in estrogen-exposed mouse ovarian tumors, and the mice exposed to estradiol had significantly diminished survival compared to those receiving a placebo pellet. Combined with the fact that epigenetic deregulation and DNA repair processes play a key role in tumorigenesis, these results suggest that in addition to fertility defects, TAF4B could also affect ovarian tumorigenesis later in life. Our preliminary data suggest that the loss of oocytes in Taf4b -knockout ovaries may occur due to deficiencies in epigenetic regulation and/or a deficiency in DNA repair during meiotic prophase, since DNA repair and meiosis related genes are significantly reduced in Taf4b -knockout ovaries. Future studies will determine if estrogen treatment of neonatal Taf4b -knockout ovaries ameliorates these epigenetic and DNA repair deficits, leading to the observed oocyte rescue, and will explore if ovarian tumorigenesis is altered in the absence of TAF4B. Citation Format: Jennifer R. Wardell, Kathryn J. Grive, Kendra M. Hodgkinson, April K. Binder, Kimberly A. Seymour, Lindsay A. Lovasco, Ken S. Korach, Barbara C. Vanderhyden and Richard N. Freiman. Estrogen-responsiveness of the TFIID subunit TAF4B and its potential function in ovarian cancer, epigenetic regulation and meiotic DNA repair. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A81.