NR4A1 and NR4A3 restrict HSC proliferation via reciprocal regulation of C/EBPα and inflammatory signaling.

Members of the NR4A subfamily of nuclear receptors have complex, overlapping roles during hematopoietic cell development and also function as tumor suppressors of hematologic malignancies. We previously identified NR4A1 and NR4A3 (NR4A1/3) as functionally redundant suppressors of acute myeloid leukemia (AML) development. However, their role in hematopoietic stem cell (HSC) homeostasis remains to be disclosed. Using a conditional Nr4a1/Nr4a3 knockout mouse (CDKO), we show that codepletion of NR4A1/3 promotes acute changes in HSC homeostasis including loss of HSC quiescence, accumulation of oxidative stress, and DNA damage while maintaining stem cell regenerative and differentiation capacity. Molecular profiling of CDKO HSCs revealed widespread upregulation of genetic programs governing cell cycle and inflammation and an aberrant activation of the interferon and NF-κB signaling pathways in the absence of stimuli. Mechanistically, we demonstrate that NR4A1/3 restrict HSC proliferation in part through activation of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific Cebpa enhancer and activating Cebpa transcription. In addition, NR4A1/3 occupy the regulatory regions of NF-κB-regulated inflammatory cytokines, antagonizing the activation of NF-κB signaling. Taken together, our results reveal a novel coordinate control of HSC quiescence by NR4A1/3 through direct activation of C/EBPα and suppression of activation of NF-κB-driven proliferative inflammatory responses.

Abrogation of nuclear receptors Nr4a3 and Nr4a1 leads to development of acute myeloid leukemia.

Nur77 (NR4A1) and Nor-1 (NR4A3) are highly homologous orphan nuclear receptors that regulate the transcription of overlapping target genes. The transcriptional activity of both proteins is regulated in a ligand-independent manner by cell- and stimulus-specific gene induction and protein phosphorylation. Nor-1 and Nur77 have been implicated in a variety of cellular processes, including the transduction of hormonal, inflammatory, mitogenic, apoptotic and differentiative signals. Cellular responses to these proteins suggest that they may function as homeostatic regulators of proliferation, apoptosis and differentiation, and thus may regulate cellular susceptibility to tumorigenesis. Their physiological functions, however, remain poorly understood. Here we describe a previously unsuspected function of Nor-1 and Nur77-as critical tumor suppressors of myeloid leukemogenesis. The abrogation of these proteins in mice led to rapidly lethal acute myeloid leukemia (AML), involving abnormal expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signaling. We found that downregulation of NR4A3 ( NOR-1 ) and NR4A1 ( NUR77 ) was a common feature in leukemic blasts from human AML patients, irrespective of karyotype. Thus Nor-1 and Nur77 may provide potential targets for therapeutic intervention in AML.

Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in mice.

The formation of the atherosclerotic lesion is a complex process influenced by an array of inflammatory and lipid metabolism pathways. We previously demonstrated that NR4A nuclear receptors are highly induced in macrophages in response to inflammatory stimuli and modulate the expression of genes linked to inflammation in vitro. Here we used mouse genetic models to assess the impact of NR4A expression on atherosclerosis development and macrophage polarization. Transplantation of wild-type, Nur77⁻/⁻, or Nor1⁻/⁻ null hematopoetic precursors into LDL receptor (LDLR)⁻/⁻ recipient mice led to comparable development of atherosclerotic lesions after high-cholesterol diet. We also observed comparable induction of genes linked to M1 and M2 responses in wild-type and Nur77-null macrophages in response to lipopolysaccharides and interleukin (IL)-4, respectively. In contrast, activation of the nuclear receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation of signal transductor and activator of transcription 6 (STAT6) strongly suppressed M2 responses. Recent studies have suggested that alterations in levels of Ly6C(lo) monocytes may be a contributor to inflammation and atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with decreased abundance of Ly6C(lo) monocytes, but this change was not correlated with atherosclerotic lesion development. Collectively, our results suggest that alterations in the Ly6C(lo) monocyte population and bone marrow NR4A expression do not play dominant roles in macrophage polarization or the development of atherosclerosis in mice.

Reduced NR4A gene dosage leads to mixed myelodysplastic/myeloproliferative neoplasms in mice.

The NR4A subfamily of nuclear receptors (NR4A1, NR4A2, and NR4A3) function as transcription factors that transduce diverse extracellular signals into altered gene transcription to coordinate apoptosis, proliferation, cell cycle arrest, and DNA repair. We previously discovered that 2 of these receptors, NR4A1 and NR4A3, are potent tumor suppressors of acute myeloid leukemia (AML); they are silenced in human AML, and abrogation of both genes in mice leads to rapid postnatal development of AML. Reduced expression of NR4As is also a common feature of myelodysplastic syndromes (MDSs). Here we show that reduced gene dosage of NR4A1 and NR4A3 in hypoallelic (NR4A1(+/-)NR4A3(-/-) or NR4A1(-/-)NR4A3(+/-)) mice below a critical threshold leads to a chronic myeloid malignancy that closely recapitulates the pathologic features of mixed myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) with progression to AML in rare cases. Enhanced proliferation and excessive apoptosis of hematopoietic stem cells and myeloid progenitors, together with elevated DNA damage, contribute to MDS/MPN disease. We identify the myeloid tumor suppressor genes Egr1 and JunB and the DNA damage checkpoint kinase, polo-like kinase 2 (Plk2) as deregulated genes whose disrupted signaling probably contributes to MDS/MPN. These mice provide a novel model to elucidate the molecular pathogenesis of MDS/MPN and for therapeutic evaluation.

Deficiency of the NR4A orphan nuclear receptor NOR1 decreases monocyte adhesion and atherosclerosis.

RATIONALE: The orphan nuclear receptor NOR1 is a member of the evolutionary highly conserved and ligand-independent NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily have been characterized as early response genes regulating essential biological processes including inflammation and proliferation; however, the role of NOR1 in atherosclerosis remains unknown. OBJECTIVE: The goal of the present study was to determine the causal contribution of NOR1 to atherosclerosis development and to identify the mechanism by which this nuclear receptor participates in the disease process. METHODS AND RESULTS: In the present study, we demonstrate expression of NOR1 in endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through a nuclear factor kappaB-dependent transactivation of the NOR1 promoter. Overexpression of NOR1 in human endothelial cells increased the expression of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule-1, whereas NOR1 deficiency altered adhesion molecule expression in response to inflammatory stimuli. Transient transfection experiments and chromatin immunoprecipitation assays revealed that NOR1 induces VCAM-1 promoter activity by binding to a canonical response element for NR4A receptors in the VCAM-1 promoter. Further functional studies confirmed that NOR1 mediates monocyte adhesion by inducing VCAM-1 and intercellular adhesion molecule-1 expression in endothelial cells. Finally, we demonstrate that NOR1 deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE(-/-) mice by decreasing the macrophage content of the lesion. CONCLUSIONS: In concert, these studies identify a novel pathway underlying monocyte adhesion and establish that NOR1 serves a previously unrecognized atherogenic role in mice by positively regulating monocyte recruitment to the vascular wall.

Deficiency of the NR4A neuron-derived orphan receptor-1 attenuates neointima formation after vascular injury.

BACKGROUND: The neuron-derived orphan receptor-1 (NOR1) belongs to the evolutionary highly conserved and most ancient NR4A subfamily of the nuclear hormone receptor superfamily. Members of this subfamily function as early-response genes regulating key cellular processes, including proliferation, differentiation, and survival. Although NOR1 has previously been demonstrated to be required for smooth muscle cell proliferation in vitro, the role of this nuclear receptor for the proliferative response underlying neointima formation and target genes trans-activated by NOR1 remain to be defined. METHODS AND RESULTS: Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in NOR1(-/-) mice compared with wild-type mice. In vitro, NOR1-deficient smooth muscle cells exhibit decreased proliferation as a result of a G(1)-->S phase arrest of the cell cycle and increased apoptosis in response to serum deprivation. NOR1 deficiency alters phosphorylation of the retinoblastoma protein by preventing mitogen-induced cyclin D1 and D2 expression. Conversely, overexpression of NOR1 induces cyclin D1 expression and the transcriptional activity of the cyclin D1 promoter in transient reporter assays. Gel shift and chromatin immunoprecipitation assays identified a putative response element for NR4A receptors in the cyclin D1 promoter, to which NOR1 is recruited in response to mitogenic stimulation. Finally, we provide evidence that these observations are applicable in vivo by demonstrating decreased cyclin D1 expression during neointima formation in NOR1-deficient mice. CONCLUSIONS: These experiments characterize cyclin D1 as an NOR1-regulated target gene in smooth muscle cells and demonstrate that NOR1 deficiency decreases neointima formation in response to vascular injury.

Progesterone receptors and ovulation.

The steroid hormone, progesterone, plays a critical role in the regulation of female ovulation. The physiological effects of progesterone are mediated by two nuclear receptor transcription factors, PR-A and PR-B, which are produced from a single gene and upon binding progesterone regulate the expression of specific gene networks in reproductive tissues. Both null mutation of the PR gene to delete both receptor proteins and selective disruption of the PR-A isoform lead to a failure of ovulation due to disabled follicular rupture in response to gonadotrophin stimulation. Recent studies have revealed that the LH stimulus that triggers ovulation is transduced by PRs residing in mural granulosa cells that induce expression of paracrine signals that interact with cumulus cells to control cumulus matrix function and expansion to facilitate follicular rupture.

Targeting Oncogenic Super Enhancers in MYC-Dependent AML Using a Small Molecule Activator of NR4A Nuclear Receptors.

Epigenetic reprogramming in Acute Myeloid Leukemia (AML) leads to the aberrant activation of super enhancer (SE) landscapes that drive the expression of key oncogenes, including the oncogenic MYC pathway. These SEs have been identified as promising therapeutic targets, and have given rise to a new class of drugs, including BET protein inhibitors, which center on targeting SE activity. NR4A nuclear receptors are tumor suppressors of AML that function in part through transcriptional repression of the MYC-driven oncogenic program via mechanisms that remain unclear. Here we show that NR4A1, and the NR4A inducing drug dihydroergotamine (DHE), regulate overlapping gene expression programs in AML and repress transcription of a subset of SE-associated leukemic oncogenes, including MYC. NR4As interact with an AML-selective SE cluster that governs MYC transcription and decommissions its activation status by dismissing essential SE-bound coactivators including BRD4, Mediator and p300, leading to loss of p300-dependent H3K27 acetylation and Pol 2-dependent eRNA transcription. DHE shows similar efficacy to the BET inhibitor JQ1 at repressing SE-dependent MYC expression and AML growth in mouse xenografts. Thus, DHE induction of NR4As provides an alternative strategy to BET inhibitors to target MYC dependencies via suppression of the AML-selective SE governing MYC expression.

Drug targeting of NR4A nuclear receptors for treatment of acute myeloid leukemia.

NR4As are AML tumor suppressors that are frequently silenced in human acute myeloid leukemia (AML). Despite their potential as novel targets for therapeutic intervention, mechanisms of NR4A silencing and strategies for their reactivation remain poorly defined. Here we show that NR4A silencing in AML occurs through blockade of transcriptional elongation rather than epigenetic promoter silencing. By intersection of NR4A-regulated gene signatures captured upon acute, exogenous expression of NR4As in human AML cells with in silico chemical genomics screening, we identify several FDA-approved drugs including dihydroergotamine (DHE) that reactivate NR4A expression and regulate NR4A-dependent gene signatures. We show that DHE induces NR4A expression via recruitment of the super elongation complex to enable elongation of NR4A promoter paused RNA polymerase II. Finally, DHE exhibits AML selective NR4A-dependent anti-leukemic activity in cytogenetically distinct human AML cells in vitro and delays AML progression in mice revealing its potential as a novel therapeutic agent in AML.

Reprogramming of the estrogen responsive transcriptome contributes to tamoxifen-dependent protection against tumorigenesis in the p53 null mammary epithelial cells.

The tumor suppressor gene p53 is frequently mutated in human breast cancer and is a marker for poor prognosis and resistance to chemotherapy. Transplantation of p53 null mouse mammary epithelium into syngeneic wild-type mice leads to normal mammary gland development followed by spontaneous mammary tumors that recapitulate many of the phenotypic, molecular and genetic features of human breast cancer. Transient exposure of p53 null mice to the anti-estrogen, tamoxifen leads to sustained and robust protection against tumor development. However the mechanism underlying this anti-tumor activity remains poorly understood. Here we demonstrate that transient exposure to tamoxifen leads to a reduction in mammary ductal side-branching and epithelial cell proliferation after tamoxifen withdrawal. Global gene expression analysis showed that transient tamoxifen exposure leads to persistent changes in the expression of a subset of estrogen regulated gene signatures in mammary epithelial cells (MECs). Among these was the protein tyrosine phosphatase, non-receptor type 5 (Ptpn5). We show that Ptpn5 is a novel tamoxifen regulated target gene which is upregulated in MECs after transient tamoxifen exposure and displays tumor suppressor activity in human breast cancer cells. Further, PTPN5 expression is strongly associated with good clinical outcome in tamoxifen treated human breast cancer patients suggesting that PTPN5 may represent a novel biomarker of tamoxifen response in human breast cancer.

nor-1 regulates hippocampal axon guidance, pyramidal cell survival, and seizure susceptibility.

The nuclear receptor transcription factor, nor-1, is expressed during mammalian development predominantly in the nervous system and is induced in a cell-specific manner in nonneuronal cells in response to a variety of extracellular stimuli. To elucidate the essential developmental functions of this transcription factor, we have analyzed the consequences of its elimination on central nervous system development in mice. Here we show that null mutant mice lacking nor-1 respond with increased limbic seizure activity to the excitotoxic glutamate receptor agonist kainic acid. We demonstrate that these abnormalities are associated with defective postnatal hippocampal development exemplified by abnormal axonal guidance of dentate gyrus granule and mossy cells, disorganization of the pyramidal cell layer, and early postnatal death of pyramidal neurons in the CA1 field of the hippocampus. Our data indicate that nor-1 plays a critical role in neuronal survival and axonal guidance in the developing murine hippocampus and that hippocampal dysgenesis in nor-1-/- mice may be an underlying cause of seizure susceptibility.

The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of the mouse inner ear.

Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of Nor-1 in mammalian development has not been established. However, recent studies indicate a function for this transcription factor in oncogenesis and apoptosis. To examine the spatiotemporal expression pattern of Nor-1 and the developmental and physiological consequences of Nor-1 ablation, Nor-1-null mice were generated by insertion of the lacZ gene into the Nor-1 genomic locus. Disruption of the Nor-1 gene results in inner ear defects and partial bidirectional circling behavior. During early otic development, Nor-1 is expressed exclusively in the semicircular canal forming fusion plates. After formation of the membranous labyrinth, Nor-1 expression in the vestibule is limited to nonsensory epithelial cells localized at the inner edge of the semicircular canals and to the ampullary and utricular walls. In the absence of Nor-1, the vestibular walls fuse together as normal; however, the endolymphatic fluid space in the semicircular canals is diminished and the roof of the ampulla appears flattened due to defective continual proliferative growth of the semicircular canals.

Iron status in mice carrying a targeted disruption of lactoferrin.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO(-/-)) mice by homologous gene targeting. LFKO(-/-) mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO(-/-) intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO(-/-) mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

Differential response of progesterone receptor isoforms in hormone-dependent and -independent facilitation of female sexual receptivity.

Neurobehavioral effects of progesterone are mediated primarily by its interaction with neural progesterone receptors (PRs), expressed as PR-A and PR-B protein isoforms. Whereas the expression of two isoforms in the neural tissues is suggestive of their selective cellular responses and modulation of distinct subsets of PR-induced target genes, the role of individual isoforms in brain and behavior is unknown. We have previously demonstrated a critical role for PRs as transcriptional mediators of progesterone (ligand-dependent), and dopamine (ligand-independent)-facilitated female reproductive behavior in female mice lacking both the isoforms of PR. To further elucidate the selective contribution of the individual PR isoforms in female sexual receptive behavior, we used the recently generated PR-A and PR-B isoform-specific null mutant mice. We present evidence for differential responses of each isoform to progesterone and dopamine agonist, SKF 81297 (SKF), and demonstrate a key role for PR-A isoform in both hormone-dependent and -independent facilitation of sexual receptive behavior. Interestingly, whereas both the isoforms were essential for SKF-facilitated sexual behavior, PR-A appeared to play a more important role in the 8-bromo-cAMP-facilitated lordosis response, raising the possibility of distinct intracellular signaling pathways mediating the responses. Finally, we also demonstrate that antiprogestin, RU38486, was an effective inhibitor of PR-A-mediated, progesterone-dependent, but not SKF or 8-bromo-cAMP-dependent sexual receptivity. The data reveal the selective contributions of individual isoforms to the signaling pathways mediating female reproductive behavior.

Progesterone receptor isoforms A and B differentially regulate MUC1 expression in uterine epithelial cells.

MUC1 expression responds differently to changes in progesterone (P) levels in mouse vs. human uterine epithelium. Two isoforms of progesterone receptor, PRA and PRB, mediate the physiological effects of P. Using transient transfection of a human uterine epithelial cell line, HEC-1A, we showed that liganded PRB stimulated MUC1 gene activity. PRA alone had little effect on MUC1 promoter activity, but antagonized the PRB-mediated stimulation. The region from 523 to 570 bp upstream of the transcriptional start site was shown to be required for the P response. Mutation of two potential P-responsive element (PRE) half-sites in this region partially inhibited the PRB-mediated response, and one PRE half-site disrupted binding of both PRB and PRA to a consensus PRE in an EMSA. These along with other studies indicated that multiple cis elements in the -523- to -570-bp region cooperate to mediate P responsiveness, and that PR interaction with other transcription factors in this region is likely. Using ovariectomized wild-type, PR knockout (PRKO), PRAKO, and PRBKO mice, P antagonism of estrogen-stimulated Muc1 protein and mRNA expression was shown to be dependent on PRA. In summary, these data show that liganded PRB stimulates MUC1 expression in human uterine epithelial cells, whereas liganded PRA antagonizes MUC1 expression in both human and mouse uterine epithelial cells. The differential MUC1 response to P in these two species may be due to dissimilar expression of the two PR isoforms in the uterine epithelium.

Nuclear receptor NR4A1 is a tumor suppressor down-regulated in triple-negative breast cancer.

The nuclear receptor (NR) superfamily contains hormone-inducible transcription factors that regulate many physiological and pathological processes through regulating gene expression. NR4A1 is an NR family member that still does not have an identified endogenous ligand, and its role in cancer is also currently unclear and controversial. In this study, we aimed to define the expression profiles and specific role of NR4A1 in the highly malignant triple-negative breast cancer (TNBC), which still lacks available targeted therapies. Bioinformatic analysis revealed a decrease of NR4A1 mRNA expression in human TNBC samples. Semi-quantitative analysis of NR4A1 protein expression by immunohistochemistry also identified a progressive NR4A1 reduction during the development of mouse basal-like mammary tumors and a significant NR4A1 downregulation in human TNBC samples. Furthermore, the expression levels of NR4A1 in human TNBC were negatively associated with tumor stage, lymph node metastasis and disease recurrence. Moreover, ectopic expression of NR4A1 in MDA-MB-231, a TNBC cell line with little endogenous NR4A1, inhibited the proliferation, viability, migration and invasion of these cells, and these inhibitions were associated with an attenuated JNK1-AP-1-cyclin D1 pathway. NR4A1 expression also largely suppressed the growth and metastasis of these cell-derived tumors in mice. These results demonstrate that NR4A1 is downregulated in TNBC and restoration of NR4A1 expression inhibits TNBC growth and metastasis, suggesting that NR4A1 is a tumor suppressor in TNBC.

Overlapping and distinct expression of progesterone receptors A and B in mouse uterus and mammary gland during the estrous cycle.

In rodents, progesterone receptors (PRs) A and B have different and often nonoverlapping roles, and this study asked whether different activities of the PR proteins in mouse are related to differences in their expression in reproductive tissues. The individual expression of PRA and PRB was determined immunohistochemically in mammary gland and uterus during the estrous cycle or in response to endocrine manipulation. In the mammary gland, PRA and PRB were colocated in PR+ epithelial cells, with little change during the estrous cycle. In the uterus, PRA was not detected in luminal epithelium at any stage of the cycle, and PR+ luminal cells expressed only PRB. In the stroma and myometrium, PRA and PRB levels fluctuated with cyclical systemic hormone exposure. Observation of functional end points suggested that augmented stromal and/or myometrial PRA in proestrus inhibited estrogen receptor expression and epithelial proliferation. Colocation of PRA and PRB was hormonally regulated, and ovariectomy did not reproduce the expression of PRA and PRB in the uterus during the estrous cycle. Whereas PRB was the only PR in the luminal epithelium in cycling mice, ovariectomy restored PRA expression, resulting in PRA-PRB colocation. In stroma and myometrium, PRA and PRB colocated in PR+ cells, but ovariectomy reduced PRA levels more than PRB, resulting in PRB-only-expressing cells. This study has shown that nonoverlapping PRA and PRB expression in the uterus, in particular the lack of PRA, and expression of PRB only in the luminal epithelium throughout the estrous cycle, is likely to contribute to the distinct roles of PRA and PRB in the adult mouse.

Nitric oxide mediates increased susceptibility to dopaminergic damage in Nurr1 heterozygous mice.

Knocking out of Nurr1 gene, a member of nuclear receptor superfamily, causes selective agenesis of dopaminergic neurons in midbrain. Reduced expression of Nurr1 increases the vulnerability of mesencephalic dopamine neurons to dopaminergic toxins. We evaluated the role of nitric oxide as a possible mechanism for this increased susceptibility. Increased expression of neuronal nitric oxide synthase and increased 3-nitrotyrosine were observed in striatum of Nurr1 heterozygous (Nurr1 +/-) mice as compared with wild-type. Increased cytochrome C activation and consecutive release of Smac/DIABLO were also observed in Nurr1 +/- mice. An induction of active Caspase-3 and p53, cleavage of poly-ADP (RNase) polymerase and reduced expression of bcl-2 were observed in Nurr1 +/- mice. Methamphetamine significantly increased these markers in Nurr1 +/- mice as compared with wild-type. The present data therefore suggest that nitric oxide plays a role as a modulating factor for the increased susceptibility, but not the potentiation, of the dopaminergic terminals in Nurr1 +/- mice. We also report that this increased neuronal nitric oxide synthase expression and increased nitration in Nurr1 +/- mice led to the activation of apoptotic cascade via the differential alterations in the DNA binding activity of transcription factors responsible for the propagation of growth arrest as well as apoptosis.

Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells.

Members of the NR4A subfamily of orphan nuclear receptors regulate cell fate decisions via both genomic and non-genomic mechanisms in a cell and tissue selective manner. NR4As play a key role in maintenance of hematopoietic stem cell homeostasis and are critical tumor suppressors of acute myeloid leukemia (AML). Expression of NR4As is broadly silenced in leukemia initiating cell enriched populations from human patients relative to normal hematopoietic stem/progenitor cells. Rescue of NR4A expression in human AML cells inhibits proliferation and reprograms AML gene signatures via transcriptional mechanisms that remain to be elucidated. By intersecting an acutely regulated NR4A1 dependent transcriptional profile with genome wide NR4A binding distribution, we now identify an NR4A targetome of 685 genes that are directly regulated by NR4A1. We show that NR4As regulate gene transcription primarily through interaction with distal enhancers that are co-enriched for NR4A1 and ETS transcription factor motifs. Using a subset of NR4A activated genes, we demonstrate that the ETS factors ERG and FLI-1 are required for activation of NR4A bound enhancers and NR4A target gene induction. NR4A1 dependent recruitment of ERG and FLI-1 promotes binding of p300 histone acetyltransferase to epigenetically activate NR4A bound enhancers via acetylation at histone H3K27. These findings disclose novel epigenetic mechanisms by which NR4As and ETS factors cooperate to drive NR4A dependent gene transcription in human AML cells.

Revealing progesterone's role in uterine and mammary gland biology: insights from the mouse.

Apart from distinguishing the in vivo effects of progesterone (P) from those of estrogen (E), the progesterone receptor knockout (PRKO) mouse has furnished unprecedented access to novel cell-signaling paradigms, hitherto unsuspected. Along with providing new cellular principles by which P influences proliferative and differentiative programs obligate for tissue development and tumor progression, the PRKO in conjunction with transcript profiling has begun to uncover the transcriptional cascades underlying these processes. Moreover, studies on isoform-specific knockouts for PR-A (PR-AKO) and PR-B (PR-BKO) have clearly defined distinct physiological roles for the two subtypes of PR, providing essential physiological support for previous in vitro observations. Although the PR-AKO exhibits an infertility phenotype, the PR-BKO displays normal fecundity. Conversely, although normal mammary morphogenesis can manifest in the PR-AKO, pregnancy-associated mammary morphogenesis is severely impaired in the PR-BKO. By virtue of its ability to suppress E-induced and PR-B-mediated uterine and mammary proliferation, the PR-A isoform is likely to be an attractive drug target for the next generation of selective PR modulators in the treatment of uterine and mammary gland hyperplasia. Along with defining the dynamic interplay between E and P responses and physiological events mediated by PR-A and PR-B, further studies on these models should provide a broader conceptual framework for understanding abnormal progestin responses in vivo, with attendant implications for the management of female reproductive health and for the diagnosis and/or treatment of breast cancer.