Austin J. Cooney

P53 is a barrier to somatic cell reprogramming. Deletion or transient suppression of p53 increases the efficiency of reprogramming of somatic cells into induced pluripotent stem cells. Whether p53 represents an obstacle to a similar process transdifferentiation of somatic cells is unknown. However, it is predicted that inhibition of p53 would promote transdifferentiation of fibroblasts into cardiomyocytes. In this study, the effect of p53 on the capacity of cardiogenic transdifferentiation is evaluated using p53 wild-type (p53^+/+), p53 heterozygous mutant (p53^+/−), and p53 homozygous mutant (p53^−/−) mouse embryonic fibroblasts (MEFs). Repression of p53 in MEFs increases the expression level of mesoderm transcription factors Brachyury (T) and MESP1. The cardiac-specific markers, Myh6 (Myosin, Heavy Chain 6), Myh7 (Myosin, Heavy Chain 7), and cTnI (cardiac muscle troponin I), show elevated expression in p53^+/− and p53^−/− MEFs compared with wild-type MEFs, but cardiac muscle troponin T (cTnT) showed a lower expression level when p53 was inhibited. After induction to cardiac differentiation, cTnT expression increased and markers of endoderm and ectoderm decreased in p53^+/− and p53^−/− MEFs. The effect of an important reprogramming factor Oct4 on cardiac transdifferentiation was also evaluated in the allelic series of p53 MEFs. We found that overexpression of Oct4 significantly enhanced Mesp1, Tbx5, and Isl1 expression in p53^+/+ and p53^+/− MEFs. Oct4 also enhanced cTnT expression in all three cell lines, especially in p53^+/− MEFs. Thus, inhibition of p53 expression and viral expression of Oct4 both promote transdifferentiation of MEFs into cardiomyocytes, establishing reciprocity of action in the process.

E12009 Background: Gametogenetin binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2 has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor (NR) binding motif. It has been reported that GGNBP2 being remarkably down-regulated in several human cancer cell lines. The similar NR binding motifs are also found in some other known breast cancer susceptibility genes such asBRCA1, suggesting that GGNBP2 may function as a candidate of breast cancer susceptibility gene. Methods: A cancer expression array containing 50 pair-wise human normal and breast tumor tissues were utilized to examine GGNBP2 expression in breast cancer samples. To address the potential anti-breast tumor activity of GGNBP2 in vitro, we expressed exogenous GGNBP2 in T47D, MCF-7, MCF-10A and MCF-10F cells. Cell proliferation, migration and invasion were assessed by cell growth curve, wound healing assays and colony formation assays, respectively. In vitro protein-protein interaction and luciferase reporter assays were performed to determine whether GGNBP2 interacts with ERα and modulates its transcriptional activity. qPCR and Western blot were carried out to evaluate the effects of GGNBP2 overexpression on ERa target genes, CCND1 and TFF1 expression. The anti-oncogenic activity of GGNBP2 was further verified by xenograft in mice. Results: GGNBP2 expression is reduced in primary human breast tumors. Overexpression of GGNBP2 selectively inhibits the proliferation of ERα positive breast cancer cells. Stable GGNBP2 overexpression in T47D cells inhibits 17β-estradiol (E2)–stimulated proliferation as well as migration, invasion, anchorageindependent growth in vitro and xenograft tumor growth in mice. We further demonstrate that GGNBP2 protein physically interacts with ERα, inhibits E2-induced activation of estrogen response element-driven reporter activity and attenuates ER target gene expression in T47D cells. Conclusions: GGNBP2 is a novel breast cancer tumor suppressor functioning as a nuclear receptor co-repressor to restrain ERα activity and tumorigenesis in human breast cancer.

Orphan nuclear receptors such as germ cell nuclear factor (GCNF), steroidogenic factor 1 (SF-1) and liver receptor homolog-1 (LRH-1), are emerging as important ovarian factors in regulating female reproduction. Within the ovary, GCNF (NR6A1) expression is restricted to the oocyte, while SF-1 (NR5A1) is expressed only in the somatic cells, such as granulosa, thecal and luteal cells, and interstitial cells. LRH-1 (NR5A2), an orphan receptor closely related to SF-1, is expressed only in the granulosa cells of the follicles and luteal cells within the ovary. Recent studies using conditional knockout strategies to bypass the embryonic lethality of GCNF and SF-1 null mice have uncovered important roles of GCNF and SF-1 in the oocyte and granulosa cells, respectively. In this review, we will summarize the major findings of GCNF and SF-1 in the ovary from the studies of conditional GCNF and SF-1 knockout mice. The potential role of LRH-1 in the ovary is also briefly discussed. Understanding the ovarian functions of these orphan nuclear receptors may lead to the development of new agents for regulation of female fertility and new medicines for the treatment of female idiopathic infertility, premature ovarian failure, polycystic ovarian syndrome and ovarian cancer.

Embryonic stem (ES) cell pluripotency and differentiation are controlled by a network of transcription factors and signaling molecules. Transcription factors such as Oct4 and Nanog are required for self-renewal and maintain the undifferentiated state of ES cells. Decreases in the expression of these factors indicate the initiation of differentiation of ES cells. Inactivation of the gene encoding the orphan nuclear receptor GCNF showed that it plays an important role in the repression of Oct4 expression in somatic cells during early embryonic development. GCNF^−/− ES cells were isolated to study the function of GCNF in the down-regulation of pluripotency genes during differentiation. Loss of repression of ES cell marker genes Oct4, Nanog, Sox2, FGF4, and Stella was observed upon treatment of GCNF^−/− ES cells with retinoic acid. The loss of repression of pluripotency genes is either a direct or indirect consequence of loss of GCNF. Both the Oct4 and Nanog genes are direct targets of GCNF repression during ES cell differentiation and early mouse embryonic development. In contrast Sox2 and FGF4 are indirectly regulated by GCNF through Oct4. These findings establish a central role for GCNF in the repression of pluripotency gene expression during retinoic acid-induced ES cell differentiation.

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.

Germ cell nuclear factor (GCNF) is a member of the nuclear receptor superfamily, which is expressed in the adult predominantly in the male and female germ cells. In the male, GCNF is expressed in spermatogenic cells. GCNF binds as a homodimer to direct repeat response elements of the consensus half-site sequence, AGGTCA, with 0 bp spacing (DR0). Using this information, a search of genomic databases was performed to identify candidate GCNF responsive, spermatogenic-specific, genes that contain DR0 sequences. The mouse protamine genes are the strongest candidates identified to date, as they are post-meiotically expressed in round spermatids and contain DR0 elements in their proximal promoters. Previous work has shown that both recombinant and endogenous GCNF bind to DR0 elements in the mouse protamine 1 and 2 (Prm 1 and Prm 2) promoters with high affinity and specificity. The present work shows that in transient transfection assays in GC-1 and JEG-3 cells, co-transfection of a GCNF-VP16 expression plasmid with reporter plasmids containing either the wild type Prm 1 or Prm 2 promoter established that GCNF-VP16 is able to regulate transcription from both promoters in a DR0-dependent manner. Wild type GCNF, in contrast, acts as a repressor of basal transcription on both the Prm 1 and Prm 2 promoters in a DR0-dependent manner. Furthermore, CREMτ activation of these promoters is also repressed by wild-type GCNF, indicating that GCNF also acts as a repressor of activated transcription. GCNF therefore defines a novel nuclear receptor-signaling pathway that may regulate a subset of genes involved in the terminal differentiation process of spermatogenesis, exemplified by the protamines. Mol. Reprod. Dev. 68: 394–407, 2004.