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.

Targeting TBP-Associated Factors in Ovarian Cancer.

As ovarian tumors progress, they undergo a process of dedifferentiation, allowing adaptive changes in growth and morphology that promote metastasis and chemoresistance. Herein, we outline a hypothesis that TATA-box binding protein associated factors (TAFs), which compose the RNA Polymerase II initiation factor, TFIID, contribute to regulation of dedifferentiation states in ovarian cancer. Numerous studies demonstrate that TAFs regulate differentiation and proliferation states; their expression is typically high in pluripotent cells and reduced upon differentiation. Strikingly, TAF2 exhibits copy number increases or mRNA overexpression in 73% of high-grade serous ovarian cancers (HGSC). At the biochemical level, TAF2 directs TFIID to TATA-less promoters by contact with an Initiator element, which may lead to the deregulation of the transcriptional output of these tumor cells. TAF4, which is altered in 66% of HGSC, is crucial for the stability of the TFIID complex and helps drive dedifferentiation of mouse embryonic fibroblasts to induced pluripotent stem cells. Its ovary-enriched paralog, TAF4B, is altered in 26% of HGSC. Here, we show that TAF4B mRNA correlates with Cyclin D2 mRNA expression in human granulosa cell tumors. TAF4B may also contribute to regulation of tumor microenvironment due to its estrogen-responsiveness and ability to act as a cofactor for NFκB. Conversely, TAF9, a cofactor for p53 in regulating apoptosis, may act as a tumor suppressor in ovarian cancer, since it is downregulated or deleted in 98% of HGSC. We conclude that a greater understanding of mechanisms of transcriptional regulation that execute signals from oncogenic signaling cascades is needed in order to expand our understanding of the etiology and progression of ovarian cancer, and most importantly to identify novel targets for therapeutic intervention.

Accelerated ovarian aging in the absence of the transcription regulator TAF4B in mice.

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.

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.

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection.

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5'-TCCRAC-3'. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5'-GTYGGA-3'. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.

Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b.

Oocyte development in the mammalian ovary requires productive interactions with somatic granulosa cells of the ovarian follicle. Proliferating granulosa cells support the progression of follicular growth and maturation, multiplying dramatically as it unfolds. The cell cycle recruitment of granulosa cells is regulated at least in part by hormones such as follicle-stimulating hormone (FSH) and estrogen. Follicles recruited into the growth phase following formation of multiple layers of granulosa cells have two major fates: either to continue proliferation followed by differentiation, or to die by programmed cell death, or atresia. While many of the signaling pathways orchestrating ovarian follicle development are known, the downstream transcriptional regulators that integrate such signals in the mammalian ovary remain to be defined. Recent experiments in diverse organisms have revealed multiple instances of gonad-selective components of the basal transcriptional machinery. One such protein, TAF4b, is a gonadal-enriched coactivator subunit of the TFIID complex required for normal female fertility in the mouse. To determine the etiology of female infertility of the TAF4b-deficient mice, we have determined multiple functions of TAF4b during postnatal ovarian follicle development. Here we demonstrate that the TAF4b protein is expressed in the granulosa cell compartment of the mammalian ovarian follicle. Furthermore, TAF4b-deficient mouse ovaries contain reduced numbers of primordial as well as growing follicles and a concomitant increased proportion of apoptotic follicles in comparison to wild type counterparts. Importantly, TAF4b-null follicles are largely resistant to induction of proliferation in response to multiple hormonal stimuli including estrogen and FSH and demonstrate compromised granulosa cell survival. Together, these data suggest that TAF4b integrates a program of granulosa cell gene expression required for normal ovarian follicle survival and proliferation in response to diverse ovarian signaling events.