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.

Detection of PACH1, a nuclear factor implicated in the transcriptional regulation of meiotic and early haploid stages of spermatogenesis.

Spermatogenesis occurs in a series of well-defined stages and serves as an excellent model for lineage-specific cell development. Yet, little is known regarding the transcriptional mechanisms responsible for cell- and stage-dependent gene regulation in the male germ line. The rat and mouse proenkephalin genes are expressed from an alternative, spermatogenic cell-specific promoter specifically in meiotically-active pachytene spermatocytes and early post-meiotic spermatids. This promoter thus serves as an excellent model for defining transcriptional regulators involved in germ line-specific gene expression in meiotic cells. Previous transgenic studies identified a proximal, 51 bp 5'-flanking sequence containing two direct repeat elements that are absolutely required for in vivo proenkephalin promoter activity in spermatocytes and spermatids. Here, footprinting analyses were used to further delineate the specific interactions of a spermatogenic cell nuclear factor with the repeat elements within the proximal promoter region. This repeat-binding factor was also shown to be developmentally upregulated specifically in pachytene spermatocytes. Using Southwestern analysis, we have identified a unique nuclear protein enriched in pachytene spermatocytes that specifically recognizes the repeat elements within the proximal 5'-flanking sequence. We propose that this DNA binding factor, termed PACH1, is a key transcriptional regulator of the proenkephalin and potentially other gene promoters, uniquely expressed during meiosis in the male germ line.

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.

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.

The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.

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.

Proenkephalin gene expression in testicular interstitial cells is down-regulated coincident with the appearance of pachytene spermatocytes.

Two distinct forms of proenkephalin messenger RNA (mRNA) are present in the murine testis, a family of 1.7 kilobases (kb), germ cell-specific transcripts and a 1.45-kb form that is also found in somatic tissues. In situ hybridization and molecular analysis of purified spermatogenic cell types were used to characterize the cellular localization of these different transcripts during development of the mouse testis. Both forms of proenkephalin mRNA were observed in isolated germ cells by RNA gel-blot analysis, but in distinct developmental patterns; the 1.7-kb transcripts were present in cells undergoing meiosis and spermiogenesis, whereas the 1.45-kb mRNA was detected primarily in type B spermatogonia. In contrast, in situ hybridization analysis did not detect significant amounts of the 1.45-kb transcript in any spermatogenic cell type. Using transcript-specific probes, distinct patterns of developmental expression were evident for the two mRNAs. The 1.45-kb transcript was the only form detected in the prepubertal testis, where it was localized mainly in interstitial cells. In contrast, the 1.7-kb transcripts were the major mRNAs observed in the adult testis and were localized to spermatogenic cells. A transition from the prepubertal to the adult pattern occurred on or about postnatal day 21, when proenkephalin-expressing pachytene spermatocytes begin to populate the seminiferous tubules. In situ hybridization analysis further demonstrated that proenkephalin gene expression in mutant (at/at) mice, which lack germ cells, was identical to that observed in the early prepubertal testis. These results suggest that the 1.45-kb proenkephalin mRNA is developmentally down-regulated in mouse interstitial cells and that this process requires ongoing spermatogenesis.

The rat proenkephalin germ line promoter contains multiple binding sites for spermatogenic cell nuclear proteins.

Rat and mouse spermatogenic cells contain a family of 1700-nucleotide (nt) proenkephalin mRNAs that are generated from an alternate, germ cell-specific promoter. This promoter is located approximately 350 base pairs (bp) downstream of the promoter used in somatic cells, within the first intron for the somatic transcript. In a previous study, rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing both promoters were shown to be transcribed selectively from the germ cell promoter and in the correct developmental pattern in spermatogenic cells of transgenic mice. In the present study it was found that spermatogenic cell-specific transgene expression was maintained after deletion of the upstream somatic promoter. This result establishes that the rat proenkephalin germ-line promoter is capable of functioning independently of transcriptional elements associated with the somatic promoter and localizes the requisite spermatogenic cell cis-elements to a 500-bp region encompassing the germ cell initiation sequences. A comprehensive analysis of binding sites for rat spermatogenic cell nuclear factors within this 500-bp region was performed using gel-shift and DNAse I footprinting techniques. Eight distinct binding regions were identified, each of which formed one or more cell-specific complexes with nuclear proteins from rat spermatogenic cells. These results suggest that multiple cis-acting elements may cooperate in the cell-specific and developmental regulation of rat proenkephalin gene transcription during spermatogenesis.

Selective transcription of rat proenkephalin fusion genes from the spermatogenic cell-specific promoter in testis of transgenic mice.

The rat and mouse proenkephalin genes each contains two distinct promoters, one of which is utilized exclusively by spermatogenic cells. The germ cell-specific promoter lacks TATA sequences, is G+C rich, and contains multiple initiation sites. To investigate the nature of the cis-acting elements that determine selective transcription of the proenkephalin gene in male germ cells, two rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing the two different promoter regions as well as 1.6 or 0.3 kilobases, respectively, of 5'-flanking sequence were expressed in transgenic mice. Multiple transgenic lines were developed which expressed the fusion genes in testis, brain, and heart but not in tissues that do not normally express the proenkephalin gene. Fusion gene transcripts in transgenic mouse testes were localized to those spermatogenic cell types that utilize the spermatogenic cell promoter and were selectively and accurately initiated from the multiple rat germ cell start sites. Transgenic mice thus provide a useful model for the localization and characterization of cis-acting elements mediating transcription of the proenkephalin gene from its germ cell-specific promoter.

Proenkephalin products are stored in the sperm acrosome and may function in fertilization.

Previous studies have shown that spermatogenic cells are a major source of testicular RNA encoding the opioid peptide precursor proenkephalin, suggesting that proenkephalin-derived peptides may function as intratesticular paracrine factors produced by male germ cells. However, direct evidence for the production of proenkephalin by spermatogenic cells has been lacking. In this report, we have used polysome profile analysis, peptide quantitation, and immunocytochemistry to show that proenkephalin products are synthesized during spermatogenesis and are retained within spermatozoa of humans, hamsters, rats, and sheep. We further show that these peptides are stored in the sperm acrosome and are depleted from sperm following the acrosome reaction, an exocytotic event required for fertilization. Proenkephalin products thus may serve a dual function as sperm acrosomal factors released during the fertilization process as well as intratesticular regulators secreted by spermatogenic cells.

Transcription of the rat and mouse proenkephalin genes is initiated at distinct sites in spermatogenic and somatic cells.

During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.

Widespread organ expression of the rat proenkephalin gene during early postnatal development.

The opioid peptides have been implicated as potential regulators of cell development in nervous and reproductive tissues. A survey of proenkephalin gene expression during rat development showed that the mRNA for this opioid precursor is present at substantial concentrations in several developing tissues (kidney, liver, skin, skeletal muscle, and lung) that have essentially undetectable levels in adults. In neonatal rats, skeletal muscle has greater concentrations of this transcript than brain. Polysomal analysis further demonstrated that proenkephalin mRNA is actively translated in skeletal muscle from newborn rats. These results raise the possibility that proenkephalin and its products perform a general regulatory role in cell proliferation or differentiation.

Translational status of proenkephalin mRNA in the rat reproductive system.

The mRNA for the opioid peptide precursor proenkephalin is widely distributed throughout the male and female reproductive systems of rodents. In the present studies, the concentrations of proenkephalin-derived peptides in selected reproductive tissues of the rat have been determined. When compared with previously characterized tissues such as brain, the peptide contents in reproductive tissues were unexpectedly low relative to the abundance of proenkephalin mRNA. This suggested that either translation of proenkephalin mRNA is relatively inefficient in reproductive tissues or that the turnover of proenkephalin products occurs at a higher rate, or both. To distinguish between these possible mechanisms, the polysomal distributions of proenkephalin mRNA in different rat reproductive tissues and in rat brain were determined. In adult rat testis, in which the predominant proenkephalin RNA is the 1700-nucleotide form present in spermatogenic cells, the transcript was found to be mainly associated with translationally inactive ribonucleoprotein fractions. In contrast, the 1450-nucleotide form of proenkephalin mRNA appeared to be translated to a similar extent in rat brain, epididymis, ovary, and somatic cells of the immature rat testis. It therefore appears that inefficient translation of proenkephalin mRNA in spermatogenic cells is a major determinant of the low ratio of proenkephalin peptides to RNA in the adult rat testis, while posttranslational mechanisms (most likely peptide turnover) are involved in the rat epididymis, ovary, and presumably other reproductive tissues. These findings also indicate that mRNA and/or translation product concentrations within a given tissue are not always accurate indicators of the level of peptide or protein production.