Daniel L. Kilpatrick

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 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.

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-τ 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.

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.

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 accompained 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.

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.

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.

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. Mol. Reprod. Dev. 57:224–231, 2000.

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. Mol. Reprod. Dev. 46:476–481, 1997.