DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation.

Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program.

Nuclear receptor-induced chromosomal proximity and DNA breaks underlie specific translocations in cancer.

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

The instructive role of metanephric mesenchyme in ureteric bud patterning, sculpting, and maturation and its potential ability to buffer ureteric bud branching defects.

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 (E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-P(i) type 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a "branchless" UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFRalpha1 (a receptor for GDNF) to tips. Such "quality control" by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-dependent gene networking in interchromatin granules.

Although the role of liganded nuclear receptors in mediating coactivator/corepressor exchange is well-established, little is known about the potential regulation of chromosomal organization in the 3-dimensional space of the nucleus in achieving integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among specific subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories, which depends on the actions of nuclear actin/myosin-I machinery and dynein light chain 1. The histone lysine demethylase, LSD1, is required for these ligand-induced interactive loci to associate with distinct interchromatin granules, long thought to serve as "storage" sites for the splicing machinery, some critical transcription elongation factors, and various chromatin remodeling complexes. We demonstrate that this 2-step nuclear rearrangement is essential for achieving enhanced, coordinated transcription of nuclear receptor target genes.

TBL1 and TBLR1 phosphorylation on regulated gene promoters overcomes dual CtBP and NCoR/SMRT transcriptional repression checkpoints.

A key strategy to achieve regulated gene expression in higher eukaryotes is to prevent illegitimate signal-independent activation by imposing robust control on the dismissal of corepressors. Here, we report that many signaling pathways, including Notch, NF-kappaB, and nuclear receptor ligands, are subjected to a dual-repression "checkpoint" based on distinct corepressor complexes. Gene activation requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer beta-like proteins TBL1 and TBLR1, that license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, their function and differential specificity reside in only five specific Ser/Thr phosphorylation site differences, regulated by direct phosphorylation at the level of the promoter, as exemplified by the role of PKCdelta in TBLR1-dependent dismissal of NCoR. Thus, our data reveal a strategy of dual-factor repression checkpoints, in which dedicated exchange factors serve as sensors for signal-specific dismissal of distinct corepressors, with specificity imposed by upstream signaling pathways.

The role of GRASP55 in Golgi fragmentation and entry of cells into mitosis.

GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.

Nuclear receptor-enhanced transcription requires motor- and LSD1-dependent gene networking in interchromatin granules.

While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.

Atbf1 is required for the Pit1 gene early activation.

Enhancers have been functionally described for >35 years, but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood. Using a combination of in vivo enhancer mapping and proteomics approaches, we have established that two distant and distinct early enhancers, each requiring different transcription complexes, are required for full activation of the gene encoding the pituitary lineage determining factor, Pit1. A transcription factor belonging to the "giant, multiple-homeodomain and zinc finger family," Atbf1, serves as a novel pituitary regulator for one of the two required enhancers as shown by genetic and in vitro analysis.

Robert A. Good, the March of Dimes, and immunodeficiency: an historical perspective.

Dr. Robert A. Good and the March of Dimes Birth Defects Foundation maintained a close association for a quarter century in the fight against immunodeficiency diseases. The March of Dimes, whose mission is to prevent birth defects, premature birth, and infant mortality, awarded an initial grant to Dr. Good in 1960 to conduct basic clinical and experimental studies on arthritis and collagen diseases. By 1966, this support broadened to include Dr. Good's research on agammaglobulinemia, ataxia telangiectasia, Chediak-Higashi disease, and Wiskott-Aldrich syndrome. Dr. Good led three historic March of Dimes conferences on immunodeficiency and, in 1968, conducted the first bone marrow transplant to correct an immunological birth defect, memorialized by the March of Dimes in its educational film, Decision (1970). March of Dimes grants to Dr. Good for his research in cellular engineering to genetically correct the defined birth defects approached $1 million for the period 1960-1985.

Differential repression of c-myc and cdc2 gene expression by ERF and PE-1/METS.

The molecular mechanisms that control the proliferation and differentiation of specific cell types remain poorly understood. Positive ETS factors play important roles in mediating proliferative responses to Ras/MAPK signaling in many cell types following mitogenic stimulation. PE-1/METS, a member of the ETS-domain family transcription factors that functions as a transcriptional repressor, can block mitogenic responses mediated by positively acting Ets factors. The anti-proliferative functions of PE-1/METS require its interaction with DP103, a multifunctional DEAD-box protein that mediates interactions with corepressor proteins and acts in a cooperative manner with Rb family members and to repress cell cycle control genes. ETS-2 repressor factor (ERF) is structurally related to and also functions as a transcriptional repressor, but endogenous target genes and mechanisms of repression remain unknown. Here, we demonstrate that like PE-1/METS, ERF-mediated repression also requires DP103, and that ERF negatively regulates the c-myc and cdc2 genes. In contrast to PE-1/METS, however, ERF-mediated repression of these genes is inactivated by MAPK signaling through phosphorylation sites that are ERF-specific. Furthermore, constitutive activation of the Ras/MAPK pathway in RAW 264.7 cells transformed by the v-Abelson leukemia virus is associated with constitutive inactivation of ERF in this cell type. We propose that ERF and PE-1/METS function to impose 'repression checkpoints' on a subset of cell cycle control genes that are differentially regulated by growth factor signaling pathways that control proliferation and differentiation and that ERF is targeted for inactivation by transforming oncogenes such as vAbl.

Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors.

Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

Regulation of NF-kappaB-dependent gene expression by the POU domain transcription factor Oct-1.

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.

Genetic manipulation of Mammalian cells by microinjection.

INTRODUCTIONMicroinjection allows the introduction of molecules into a defined population of cells at a defined concentration, and the timing of the experiment can be controlled stringently, minimizing problems associated with overexpression. Delivery by microinjection can be used for any type of cell that is adherent in culture, including primary cells. Perhaps its most powerful aspect is the ability to introduce several types of reagents into cells simultaneously, including DNA constructs, a labeled dextran to mark injected cells, antibodies, short interfering RNAs (siRNAs), and peptides. Because siRNA can be generated easily and rapidly for any target gene, it is relatively simple to assess many effects of the knockout of any gene in any type of adherent cell in a matter of days. However, microinjection is not trivial to learn, and requires attention to detail. Also, the limited number of cells involved often does not permit the subsequent analysis of effects on some biochemical parameters. While the major limitation of the approach is the small amount of material obtained, as the sensitivity of analysis techniques increases, so does the usefulness of this method. We emphasize here recent advances in the use of microinjection in combination with siRNA technology.

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system.

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.

A topoisomerase IIbeta-mediated dsDNA break required for regulated transcription.

Multiple enzymatic activities are required for transcriptional initiation. The enzyme DNA topoisomerase II associates with gene promoter regions and can generate breaks in double-stranded DNA (dsDNA). Therefore, it is of interest to know whether this enzyme is critical for regulated gene activation. We report that the signal-dependent activation of gene transcription by nuclear receptors and other classes of DNA binding transcription factors, including activating protein 1, requires DNA topoisomerase IIbeta-dependent, transient, site-specific dsDNA break formation. Subsequent to the break, poly(adenosine diphosphate-ribose) polymerase-1 enzymatic activity is induced, which is required for a nucleosome-specific histone H1-high-mobility group B exchange event and for local changes of chromatin architecture. Our data mechanistically link DNA topoisomerase IIbeta-dependent dsDNA breaks and the components of the DNA damage and repair machinery in regulated gene transcription.

Macrophage/cancer cell interactions mediate hormone resistance by a nuclear receptor derepression pathway.

Defining the precise molecular strategies that coordinate patterns of transcriptional responses to specific signals is central for understanding normal development and homeostasis as well as the pathogenesis of hormone-dependent cancers. Here we report specific prostate cancer cell/macrophage interactions that mediate a switch in function of selective androgen receptor antagonists/modulators (SARMs) from repression to activation in vivo. This is based on an evolutionarily conserved receptor N-terminal L/HX7LL motif, selectively present in sex steroid receptors, that causes recruitment of TAB2 as a component of an N-CoR corepressor complex. TAB2 acts as a sensor for inflammatory signals by serving as a molecular beacon for recruitment of MEKK1, which in turn mediates dismissal of the N-CoR/HDAC complex and permits derepression of androgen and estrogen receptor target genes. Surprisingly, this conserved sensor strategy may have arisen to mediate reversal of sex steroid-dependent repression of a limited cohort of target genes in response to inflammatory signals, linking inflammatory and nuclear receptor ligand responses to essential reproductive functions.

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells.

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Identification and characterization of SAP25, a novel component of the mSin3 corepressor complex.

The transcriptional corepressor mSin3 is associated with histone deacetylases (HDACs) and is utilized by many DNA-binding transcriptional repressors. We have cloned and characterized a novel mSin3A-binding protein, SAP25. SAP25 binds to the PAH1 domain of mSin3A, associates with the mSin3A-HDAC complex in vivo, and represses transcription when tethered to DNA. SAP25 is required for mSin3A-mediated, but not N-CoR-mediated, repression. SAP25 is a nucleocytoplasmic shuttling protein, actively exported from the nucleus by a CRM1-dependent mechanism. A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. An isotope-coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the mSin3 complex, nuclear export machinery, and regulators of transcription and cell cycle. These results suggest that SAP25 is a novel core component of the mSin3 corepressor complex whose subcellular location is regulated by PML.