Post-transcriptional control of candidate risk genes for type 1 diabetes by rare genetic variants.

The genetic variation causal for predisposition to type 1 diabetes (T1D) remains unidentified for the majority of known T1D risk loci. MicroRNAs function as post-transcriptional gene regulators by targeting microRNA-binding sites in the 3' untranslated regions (UTR) of mRNA. Genetic variation within the 3'-UTR of T1D-associated genes may contribute to T1D development by altering microRNA-mediated gene regulation. In silico analysis of variable sites predicted altered microRNA binding in established T1D loci. Functional implications were assessed for variable sites in the 3'-UTR of T1D candidate risk genes CTLA4 and IL10, both involved in immune regulation. We confirmed that in these genes 3'-UTR variation either disrupted or introduced a microRNA-binding site, affecting the repressive capacity of miR-302a* and miR-523, respectively. Our study points to the potential of 3'-UTR variation to affect T1D pathogenesis by altering post-transcriptional gene regulation by microRNAs.

Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus.

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 microm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.

The role of ATF/CREB family members in cell growth, survival and apoptosis.

Programmed cell death is an integral part of the mechanisms regulating tissue homeostasis. Defects in the apoptotic signaling pathway are often associated with uncontrolled cell proliferation, high mutation rate and malignant transformation. Transcription factors, such as the mammalian ATF/CREB family of transcriptional regulators, have diverse functions in controlling cell proliferation and apoptosis. One particular ATF/CREB family member, ATFx, is an anti-apoptotic factor that plays an essential role in cell survival. Current observations indicate that one mechanism by which ATFx inhibits cell death and promotes cell survival is by disrupting signal transmission from activated "death receptors" to initiator caspases. A better understanding of ATFx function should provide new insight into the processes that control apoptotic cascades.

E2F2 converts reversibly differentiated PC12 cells to an irreversible, neurotrophin-dependent state.

E2Fs play a central role in cell proliferation and growth arrest through their ability to regulate genes involved in cell cycle progression, arrest and apoptosis. Recent studies further indicate that this family of transcriptional regulators participate in cell fate/differentiation events. They are thus likely to have a prominent role in controlling the terminal differentiation process and its irreversibility. Here we have specifically examined the role of E2F2 in neuronal differentiation using a gain-of-function approach. Endogenous E2F2 increased in PC12 cells in response to nerve growth factor (NGF) and was also expressed in cerebellar granule neurons undergoing terminal differentiation. While PC12 cells normally undergo reversible dedifferentiation and cell cycle re-entry upon NGF removal, forced expression of E2F2 inhibited these events and induced apoptosis. Thus, E2F2 converted PC12-derived neurons from a reversible to a 'terminally' differentiated, NGF-dependent state, analogous to postmitotic sympathetic neurons. This contrasts with the effects of E2F4, which enhances the differentiation state of PC12 cells without affecting cell cycle parameters or survival. These results indicate that E2F2 may have a unique role in maintaining the postmitotic state of terminally differentiated neurons, and may participate in apoptosis in neurons attempting to re-enter the cell cycle. It may also be potentially useful in promoting the terminally arrested/differentiated state of tumor cells.

The neuroprotective and antiapoptotic effects of melatonin in cerebellar neurons involve glucocorticoid receptor and p130 signal pathways.

Melatonin is an endocrine factor known to affect a number of physiological functions. The present studies have demonstrated an additional activity for pineal melatonin, specifically associated with the survival and differentiation of neuroblasts. Based on experimental data several conclusions might be drawn. First, melatonin negatively regulates the expression of glucocorticoid receptor (GR) in cerebellar granule neurons. Second, downregulation of GR is associated with a marked decrease in programmed cell death of the granule neurons. Third, melatonin upregulates the expression of p130, which is an essential factor for the initiation and maintenance of neuronal development and differentiation. Thus, melatonin function in postmitotic neurons involves several regulatory pathways with partially overlapping roles. The biological implications are discussed in light of these results.

E2F4 actively promotes the initiation and maintenance of nerve growth factor-induced cell differentiation.

E2F transcription factors play a critical role in cell cycle progression through the regulation of genes required for G(1)/S transition. They are also thought to be important for growth arrest; however, their potential role in the cell differentiation process has not been previously examined. Here, we demonstrate that E2F4 is highly upregulated following the neuronal differentiation of PC12 cells with nerve growth factor (NGF), while E2F1, E2F3, and E2F5 are downregulated. Immunoprecipitation and subcellular fractionation studies demonstrated that both the nuclear localization of E2F4 and its association with the Rb family member p130 increased following neuronal differentiation. The forced expression of E2F4 markedly enhanced the rate of PC12 cell differentiation induced by NGF and also greatly lowered the rate at which cells lost their neuronal phenotype following NGF removal. Importantly, this effect occurred in the absence of any significant change in the growth regulation of PC12 cells by NGF. Further, the downregulation of E2F4 expression with antisense oligodeoxynucleotides inhibited NGF-induced neurite outgrowth, indicating an important role for this factor during PC12 cell differentiation. Finally, E2F4 expression was found to increase dramatically in the developing rat cerebral cortex and cerebellum, as neuroblasts became postmitotic and initiated terminal differentiation. These findings demonstrate that, in addition to its effects on cell proliferation, E2F4 actively promotes the neuronal differentiation of PC12 cells as well as the retention of this state. Further, this effect is independent of alterations in cell growth and may involve interactions between E2F4 and the neuronal differentiation program itself. E2F4 may be an important participant in the terminal differentiation of neuroblasts.

Multiple domains of melatonin receptor are involved in the regulation of glucocorticoid receptor-induced gene expression.

Melatonin, the principal hormone of the pineal gland, elicits potent anti-stress, anti-aging and oncostatic properties and influences various immunological and endocrinological functions. We have previously described the effects of melatonin on glucocorticoid receptors and suggested its potential influence on gene transcription. In the present study, the mechanistic basis for melatonin effects on glucocorticoid receptor (GR)-dependent gene expression was examined. Activation of the melatonin transduction pathway affects type I glucocorticoid receptor expression and reduces its transcriptional activity. Coexpression of the intact melatonin and glucocorticoid receptors with MMTV promoter construct reduced the GR transcriptional activity. N- and C-terminus deletions of melatonin receptor revealed the existence of regulatory sites mediating this process. These data identify for first time one of the molecular targets of melatonin action and suggest that melatonin signaling may involve relatively direct signal transmission from the cell surface to the nucleus.

Proximal promoter sequences mediate cell-specific and elevated expression of the favorable prognosis marker TrkA in human neuroblastoma cells.

The nerve growth factor receptor, TrkA, has a critical role in the survival, differentiation, and function of neurons in the peripheral and central nervous systems. Recent studies have demonstrated a strong correlation between abundant expression of TrkA and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and differentiation of neuroblastoma tumor cells and may be an important therapeutic target in the treatment of this disease. In the present study, we have examined the mechanistic basis for TrkA gene expression in human neuroblastoma cells. Northern blotting and nuclear run-on analyses demonstrated that transcription is a primary determinant of both cell-specific and variable expression of the TrkA gene in neuroblastoma cell lines that express it to different degrees. Cell-specific and variable transcription in neuroblastoma cells was recapitulated by transient transfection of TrkA promoter-luciferase reporter constructs, and regulatory sequences mediating these processes were localized to a 138-base pair region lying just upstream of the transcription initiation region. This neuroblastoma regulatory region formed multiple DNA-protein complexes in gel shift assays that were highly enriched in neuroblastoma cells exhibiting abundant TrkA expression. Thus, TrkA-positive neuroblastoma cells are distinguished by differential expression of putative transcription factors that ultimately may serve as targets for up-regulating TrkA expression in tumors with poor prognosis.

The DNA methyltransferase inhibitor 5-azacytidine specifically alters the expression of helix-loop-helix proteins Id1, Id2 and Id3 during neuronal differentiation.

In mammals, cytosine methylation is important for the regulation of gene expression and chromatin structure. Recently, we have found evidence indicating that the maintained DNA methyltransferase activity is critical for neuronal cell differentiation. In the present study, we have investigated the effect of the DNA methyltransferase inhibitor 5-azacytidine on gene regulation during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Expression of the helix-loop-helix proteins Id1, Id2 and Id3 was specifically reduced by NGF and this effect was blocked in 5-azacytidine-treated cells, concomitant with the inhibition of NGF-induced neuronal differentiation. Nuclear run-on and Id2 promoter analyses further demonstrated that the decreased transcription of Id proteins is at least in part dependent on the DNA methyltransferase activity. These findings indicate that Id proteins are downstream targets of the NGF transduction pathway. Moreover, these results suggest that therapeutic strategies using 5-azacytidine against certain types of tumors should be reconsidered because of the possible deleterious effects on neuronal cell function.

Gli family members are differentially expressed during the mitotic phase of spermatogenesis.

The Gli family of DNA binding proteins has been implicated in multiple neoplasias and developmental abnormalities, suggesting a primary involvement in cell development and differentiation. However, to date their specific roles and mechanisms of action remain obscure, and a drawback has been the lack of a model system in which to study their normal function. Here we demonstrate that Gli family members are differentially expressed during spermatogenesis in mice. Specifically, Gli and Gli3 mRNAs were detected in mouse germ cells, while Gli2 was not. Further, both Gli and Gli3 exhibited stage-dependent patterns of expression selectively in type A and B spermatogonia. Gli expression was somewhat higher in type B spermatogonia while the abundance of Gli3 transcripts was similar in type A and B cells. Gel-shift analyses also demonstrated the enrichment of DNA binding activity specific for the Gli target sequence in spermatogonial cells. These results indicate a selective role for Gli and Gli3 during mitotic stages of male germ cell development. Spermatogenesis may thus provide a unique opportunity to identify downstream targets and explore the normal function of Gli family proteins.

Characterization of a cDNA containing trinucleotide repeat sequences that is highly enriched in spermatogenic cells.

Trinucleotide repeat sequences have become of great interest due to their association with specific genetic disorders. Here we report the identification of a cDNA containing opa trinucleotide repeats from mouse testis, termed t-OPA. The opa repeat is contained within the longest open reading frame within the cDNA. Northern analysis demonstrated that four distinct t-OPA transcripts (1.6, 2.5, 3.6, 4.0 kilobases) are preferentially expressed in mouse and rat testis, with low expression in the pituitary, brain, and adrenal gland. Further, t-OPA RNAs were highly abundant in both pachytene spermatocytes and round spermatids and decreased in cytoplasts. Polysome profile analysis indicated that t-OPA mRNAs are translated in mouse testis with efficiencies similar to other transcripts expressed in late meiotic/early post-meiotic spermatogenic cells. These findings thus suggest a role for cell-specific mRNAs containing opa repeats during mouse spermatogenesis.

Novel repeat elements direct rat proenkephalin transcription during spermatogenesis.

The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-tau during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5'-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.

Nerve growth factor induced differentiation of neuronal cells requires gene methylation.

Cell differentiation in the nervous system is dictated by specific patterns of gene expression. We have investigated the role of gene methylation during differentiation of PC12 pheochromocytoma cells in response to nerve growth factor (NGF). Here we present evidence that NGF-induced neuronal differentiation is dependent on gene methylation and that this process is not associated with inhibition of cell cycle arrest. The DNA methylation inhibitor 5-azacytidine is able to block the neurite outgrowth of NGF-treated PC12 cells. Inhibition of neuronal differentiation is accompanied by significant changes in the protein and mRNA expression pattern of the high-affinity NGF receptor (trkA). These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation dependent on gene methylation. The results indicate that DNA methyltransferase is necessary for the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in growth factor-dependent cellular responses distinct from cell proliferation.

Transcription of the TATA binding protein gene is highly up-regulated during spermatogenesis.

TATA-binding protein (TBP) and its associated factors are required for transcriptional initiation by all three RNA polymerases, and evidence for regulation of their activities during early development has been recently reported. In the present study, we have investigated the regulation of TBP gene expression during male germ cell development. TBP mRNA was found to be increased more than 40-fold in rat and mouse testis and spermatogenic cells relative to somatic tissues. This up-regulation was stage-dependent, occurring specifically in meiotic and postmeiotic cells. Nuclear run-on analysis further demonstrated that transcription of the TBP gene was markedly elevated in the adult mouse testis relative to somatic tissue and prepuberal testis, indicating that transcriptional induction accounts, in large part, for the increased abundance of TBP mRNA in spermatogenic cells. In contrast to TBP mRNA, levels of TBP protein were elevated only 2.5-fold in mouse spermatogenic cells relative to somatic tissues. Polysome gradient analysis suggested that translational repression is an important determining factor in the unexpectedly low ratio of TBP protein-mRNA in male germ cells. These findings raise the possibility that transcriptional induction of TBP during spermatogenesis reflects a cell-specific homeostatic mechanism that maintains TBP protein concentrations at sufficiently high levels throughout male germ cell development.

Transcription factor Sp1 is expressed by three different developmentally regulated messenger ribonucleic acids in mouse spermatogenic cells.

Gene expression during spermatogenesis is highly cell- and stage-specific and involves the complex interplay of multiple developmentally regulated transcription factors. Recent evidence suggests that the DNA-binding protein Sp1 functions as an important trans-activator during cell development and differentiation. In the present study, the developmental expression of Sp1 was characterized during mouse spermatogenesis. Three distinct Sp1 transcripts were detected in mouse spermatogenic cells, each with a distinct developmental pattern; an 8.2-kilobase (kb) messenger RNA (mRNA) identical in size to the somatic mRNA expressed in spermatogonial cells, a larger mRNA approximately 8.8 kb in size present in meiotic cells, and a 2.4 kb mRNA in meiotic and postmeiotic germ cells. The 8.8- and 2.4-kb Sp1 transcripts were not observed in somatic cells and, thus, are male germ cell specific. Northern, ribonuclease protection, and RT-PCR assays revealed that the 2.4-kb Sp1 transcript is truncated in both the 5'- and 3'-untranslated regions relative to the somatic mRNA and lacks a short segment of the N-terminal coding region. Polysome analysis further indicated that these germ cell-specific Sp1 mRNAs are translated, albeit with a lower efficiency than the somatic transcript. Consistent with these results, spermatogenic cells were shown to contain approximately 9-fold lower concentrations of Sp1 proteins that are approximately the same size as the somatic form. Of particular interest, the apparent affinity of Sp1 DNA-binding activity in nuclear extracts from mouse germ cells was 5-fold greater than that in extracts from mouse somatic tissues. This may reflect the existence of mechanisms within mouse spermatogenic cells that compensate for the lower nuclear concentrations of Sp1 protein. These results suggest that cell- and stage-specific regulation of Sp1 gene expression and activity may be an important component of the mouse spermatogenic cell developmental program.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Tissue-specific modulation of rat glucocorticoid receptor binding activity by melatonin.

The effect of melatonin and 2-Iodo-melatonin on nuclear and cytosolic glucocorticoid receptors in the brain, pituitary, thymus and liver has been examined. The results indicate that both melatonin and 2-Iodo-melatonin administration is associated with marked changes in the density and the affinity of cytosolic and nuclear forms of glucocorticoid receptors. These observations are discussed in the context of a possible involvement of pineal melatonin in the mechanisms regulating the behaviour and metabolism of steroid receptors.

Selective effect of melatonin on the proliferation of lymphoid cells.

1. The present study was designed to investigate the effect of melatonin on the proliferation of normal lymphocytes and certain T-lymphomas and myelomas under in vitro conditions. 2. The results revealed that administration of 200 microM melatonin inhibited significantly the incorporation of [3H]thymidine into both normal mouse and human lymphocytes and T-lymphoblastoid cell lines. 3. On the contrary, melatonin provoked an increase of myeloma cell proliferation. 4. The influence of melatonin on hybridoma cell lines was negligible. 5. Collectively, these data demonstrated that the chief pineal indole affect selectively the processes of lymphoblastoid cell growth.

2-(125I) iodomelatonin binding sites in rat adrenals: pharmacological characteristics and subcellular distribution.

Specific binding sites for 2-[125I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin greater than 2-iodomelatonin greater than melatonin greater than 5-methoxytryptamine greater than 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear (0.76 fmol/mg protein) and mitochondrial (1.82 fmol/mg protein) subcellular fractions.

Melatonin and adrenal cortex steroid production: in vivo and in vitro studies.

The present study was designed to clarify the interaction between the pineal melatonin and adrenal cortex steroid production. Experiments with male rats under chronic stress conditions (sleep deprivation) revealed that melatonin circadian pattern was fully destroyed and daytime plasma concentration were significantly elevated. Constant illumination (2500 lux) during the nighttime was not able to suppress melatonin production in the stressed animals. Plasma concentration of corticosterone were increased in the stressed rats as well. The modulatory effect of melatonin on corticosterone and progesterone production by rat adrenals was studied in a superfusion system. During melatonin challenge progesterone secretion was two-three fold elevated with no effect on corticosterone content in the plasma samples. Pineal cytoplasmic glucocorticoid and progesterone receptors were investigated as well. A specific binding was not observed in that case. Presented data support the existence of direct communication between the pineal and adrenal glands.