Nervous System-Specific Proteins

POU family transcription factors in sensory neurons D. S. Latchman, C. L. Dent, K. A. Lillycrop and j. N. Wood* Medical Molecular Biology Unit, Department of Biochemistry, Division of Molecular Pathology, University College and Middlesex School of Medicine, The Windeyer Building, Cleveland Street, London W I P 6DB and *Sandoz Institute for Medical Research, 5 Gower Place, London WC I E 6BN, U.K. The transcription of cellular genes is regulated by specific proteins, known as transcription factors, which hind to specific DNA sequences in the gene promoter or enhancer and stimulate or repress expression of the gene [ 1 J . A number of different families of transcription factors have been defined in which the members of each family have similar 1)NA-binding domains [2-41. One such family of transcription factors is the PO17 family originally named for its founder members, Pit-1, Oct-1 and 2 and unc-86, but since shown to contain a large number of different transcription factors [ S , 61. The genes encoding the mammalian transcription factors Pit-1, Oct- 1 and Oct-2 were cloned and were found to share a common domain which was also found in the nematode gene unc-86, whose mutation affects sensory neuron development. This domain was therefore named the POU domain and was subsequently shown to be responsible for the DNA-binding ability of these proteins [7, 81. Interestingly the POU domain contains a region that has high sequence similarity to the homeobox sequence present in a number of Drosophila and mammalian regulatory proteins [9]. However, in the POIJ proteins, this I’OIJ-homeodomain is associated with a second conserved domain, known as the POU-specific domain, to form the full 150-160 amino acid POU domain. As with other transcription factors the POU proteins play a critical role in regulating the expression of specific cellular genes [ h ] .Moreover, it is likely that the role of POI7 proteins is of particular importance in the development of the nervous system in controlling the expression of genes that are specifically active in neuronal cells. Thus for example the unc-86 mutation in the nematode results in a lack of touch receptor neurons or male-specific cephalic companion neurons [ l o ] , indicating that this POU protein is required for the expression of specific genes involved in the development of these neuronal cell types. Similarly, the Drosophila POU protein CFIa Abbreviations used: DKG. dorsal root ganglia; HSV. herpes simplex virus; NGP, nerve growth factor; PCK, polymerasc chain reaction; I’OI I, Pit-Oct-Unc domain.

has been shown to play a critical role in the regulation of the dopa decarboxylase gene in specific dopaminergic neurons [ 111. This connection between POU proteins and the nervous system was reinforced by the work of He et al. [ 121 who prepared degenerate oligonucleotides corresponding to two highly conserved regions of the POU domain. These oligonucleotides were then used in a polymerase chain reaction (PCR) to amplify cDNA prepared from the mRNA of various tissues in the hope of isolating novel POU proteins expressed in these tissues. Indeed this work resulted in the isolation of three novel POU factors, Rrn-1, 2 and 3, from adult brain mKNA [ 121. In order to further probe the role of POU proteins in neuronal development we wished to apply the approach of He et al. [12] to the isolation of POU proteins expressed in a specific neuronal cell type, the mammalian sensory neuron. This neuronal cell type was of particular interest because, in the nematode, its development is critically dependent on the expression of the POU protein encoded by the unc-86 gene [ 101. In view of the difficulty in obtaining sufficient pure sensory neuron material, we used the immortalized ND7 cell line. This line is one of a series obtained by fusing postmitotic sensory neurons from rat dorsal root ganglia with the HAT-sensitive N18 mouse neuroblastoma cell line [ 131. The HAT-resistant cell lines obtained in this way express a number of sensory neuron proteins such as synaptophysin not present in the parental neuroblastoma, and particular cell lines exhibit electrophysiological responses to activators of nociceptive sensory neurons such as bradykinin and capsaicin [ 13, 141. W e therefore isolated RNA from ND7 cells and prepared cDNA from it using oligo d T primers and the enzyme reverse transcriptase. The cDNA product was then amplified by PCR using the approach of He et al. [ 121 in which amplification is carried out with degenerate oligonucleotide pools containing all possible DNA sequences capable of encoding two nine-amino-acid conserved regions of the POU domain. This amplification resulted in the appearance of a diffuse band of approximately 400 bases in size, consistent with the distance apart of

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the primers in the POU domain (Fig. 1). The resulting amplification products were cloned into the Rluescript vector and individual clones analysed both by hybridization with known POU clones and by DNA sequence analysis. The results of this analysis (Table 1) showed that the predicted amino acid sequences encoded by a number of clones (class 5) did not show any conserved amino acids characteristic of the POU Fig. I

Polymerase chain reaction of cDNA prepared from two samples of N D 7 cell RNA (tracks I and 2) with degenerate primers encoding two conserved regions of the POU domain The arrows indicate the positions of the expected 400-basepair product obtained by amplification of RNAs encoding POU proteins (400) and of unincorporated primers (P) I

domain apart from those encoded by the primers themselves. Therefore, those clones are likely to have arisen by spurious matching to the primers, a phenomenon also observed by He et al. [ 121. The remaining clones, however, did exhibit all the amino acids characteristic of POU domain proteins and fell into four classes. One class of clone (class 1) has a sequence closely related to the human [ 151 and mouse [ 161 octamer-binding protein, Oct- 1, and evidently encodes the rat homologue of this constitutively expressed protein [17]. The production of the Oct-1 protein itself in ND7 cells was confirmed by incubating ND7 cell extracts with a labelled octamer oligonucleotide in a DNA mobility shift assay [2]. As expected, a low mobility complex formed by Oct-1 binding was observed in this experiment (Fig. 2).

2

Fig. 2

D N A mobility shift assay using a labelled octamer oligonucleotide and protein extracts Extracts were prepared from two samples of proliferating ND7 cells (U) or from t w o samples of these cells following transfer to serum-free medium resulting in the cessation of cell division and differentiation t o a mature neuronal phenotype (D) The arrows indicate the positions of the octamer-binding proteins 00-1and Oa-2

u

I ”

Table I

POU factor clones isolated by polymerase chain reaction Number Class

of clones

I

4

2 3

7 I2

4

8

5

14

Comment Closely related to human [ I51 and

mouse [ I61 Oct- I

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Identical to mouse Oct-2 [ 161 Identical to mouse Brn-3 [ I61

Two amino acid differences from mouse Brn-3 [ I61 and class 3 clones Unrelated to POU factors

u

D

D

Nervous System-Specific Proteins

Interestingly the second class (class 2) of clone obtained was closely related in sequence to the human [lH] and mouse [I61 form of another octamer-binding protein, Oct-2 [ 191. The identification of these clones was of considerable interest in that this protein was initially thought to be confined to I3 cells where it plays a critical role in the regulation of immunoglobulin gene expression [20]. Subsequently, however, it was shown to be also expressed in the brain [ 12, 211 suggesting that it is present in neuronal cells as well as in B cells. In agreement with our cDNA studies we were able to detect the Oct-2 protein itself in ND7 cells by means of DNA mobility shift assays (Fig. 2; [19]). €Ience ND7 cells constitute the first example of an Oct-2-expressing neuronal cell line, allowing the functional role of Oct-2 in neuronal cells to be analysed. Indeed we have already used these cells to show that Oct-2 plays a critical role in inhibiting the activity of the herpes simplex virus immediate-early genes following infection of ND7 cells, by binding to an octamer-like sequence in the viral gene promoters [ 19]?and that it also inhibits the activity of cellular octamer-containing promoters [ 221. The two remaining groups of clones (classes 3 and 4) showed extremely strong sequence similarity to each other, differing by only two amino acids. One group (class 3) encoded the same amino acid sequence as that of mouse [ 101 and rat [ 121 Hrn-3, a novel factor isolated by He et al. [ 121 in their PCR amplification of rat brain mRNA. In view of the close similarity between the two types of clone we have isolated, we believe they represent two closely related forms of Hrn-3 which are expressed in ND7 cells. Interestingly, as noted by He et al. [ 121, Rrn-3 is closely homologous to the unc-86 gene which plays a critical role in sensory neuron development in the nematode. Hence the two forms of Rrn-3 we have detected may play a similarly critical role in mammalian sensory neurons. The PCR analysis therefore indicated that the NI>7 cell line expresses four distinct POU proteins - the octamer-binding proteins Oct-1 and Oct-2 and two distinct forms of Hrn-3. The use of ND7 cells as a means of analysing sensory neuron gene expression suffers from one serious drawback, however, in that the ND7 cells proliferate continuously in culture whereas mature sensory neurons do not divide. Hence, because of their proliferating nature, ND7 cells may express some POU proteins not present in sensory neurons themselves. In order to overcome this problem we incubated ND7 cells in serum-free medium, a treatment

which we have previously shown induces these cells to cease dividing and extend large numbers of processes giving them the appearance of mature neuronal cells [ 13, 141. W e then used extracts from proliferating and non-proliferating cells in DNA mobility shift assays using a labelled octamer oligonucleotide. As shown in Fig. 2, the differentiation of ND7 cells to a mature non-dividing neuronal phenotype had no effect on the level of Oct-2 observed in the assay. In contrast, however, the level of Oct-1 declined dramatically in the nondividing cells. This finding is of particular interest because, although Oct-1 is present in virtually all cell types 1231, its mRNA is undetectable in mature neurons in almost all regions of the adult rat brain including a number of areas where the Oct-2 mRNA is detectable [ 121. It is likely therefore that the absence of Oct-1 in non-dividing ND7 cells and mature neurons reflects their non-dividing nature rather than any specific absence of Oct-1 in cells of neuronal lineage, as it is present in proliferating ND7 cells. The presence of Oct-1 only in dividing cells would be consistent with its role in stimulating histone H2B gene transcription [24] and its putative role in DNA replication [25], both of which would clearly be required only in dividing cells. Non-dividing N1>7 cells therefore represent a good model system for studying sensory neuron function and can be grown in large amounts prior to differentiation to a non-dividing form. It is clearly necessary, however, to confirm insights obtained using these cells in primary cultures of sensory neurons where amounts are more limited. W e therefore isolated dorsal root ganglia (DRG) from adult rats and investigated the expression of each of the POU factors we had detected in ND7 cells. As expected in view of the non-dividing nature of DRG neurons, only very low levels of Oct-1 were observed in these experiments [26]. In contrast, however, high levels of Oct-2 and Hrn-3 were detectable by both in situ hybridization or PCR amplification using oligonucleotides specific for each factor [19, 261. Moreover, expression was still observed following culture for several days, in the presence of cytosine arabinoside (which kills dividing cells), confirming that expression of these factors was characteristic of mature non-dividing neurons [ 19,261. All the POU factors detected in ND7 cells are therefore also detectable in bona-fuie sensory neurons. This confirms the usefulness of ND7 cells as a model for sensory neuron function, provided that insights obtained in this way are tested using

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primary sensory neurons. Thus, once a factor has been identified using the large amounts of material available from ND7 cells, its predicted expression in sensory neurons can be readily confirmed using the more limited amounts of material available directly from DKG. Having identified the expression of specific POU proteins in primary sensory neurons it is possible to carry out further experiments that would not be possible with ND7 cells. Thus, for example, we have been unable to demonstrate any response of ND7 cells to nerve growth factor (NGF) whereas this factor is an essential survival factor for developing peripheral sensory neurons [27]. Moreover, adult sensory neurons can survive in culture without NGF, allowing the effects of this factor on gene expression to be analysed without the complications produced by cell death. W e therefore cultured adult sensory neurons for 5 days in the presence or absence of NGF and analysed the effect on the expression levels of the four POU factors. In these experiments [26] no effect of NGF was observed on the levels of Oct-1 or Rrn-3. In contrast, however, we observed a clear increase in the level of Oct-2 in the NGF-treated culture both at the mRNA level as analysed by PCR and at the protein level as assayed by DNA mobility shift assay. Hence, Oct-2 protein levels are regulated by NGF at the mKNA level whilst the other POU proteins are unaffected. The finding that NGF elevates Oct-2 levels is of particular interest in view of our identification of Oct-2 as an inhibitor of herpes simplex virus (HSV) immediate-early gene expression in sensory neurons. Thus, NGF is essential for the maintenance of HSV latency [28]. Indeed latent infection of sensory neurons by HSV was originally discovered by Cushing in 1905 [29] on the basis that cutting the trigeminal nerve (which prevents retrograde transport of NGF to the neuronal cell body) results in viral reactivation. Hence our observation that, in the absence of NGF, Oct-2 levels fall resulting in increased viral gene expression, provides a simple explanation for the role of NGF in I-ISV latency. It should be noted, however, that Oct-2 is unlikely to be presented in sensory neurons only to regulate viral infections. Rather, both Oct-2 and the two forms of Hrn-3 we have identified are likely to regulate the transcription of particular genes expressed in sensory neurons. It is hoped that the combination of cell-line and primary-culture approaches which we have used to identify and characterize these factors, will ultimately enable us

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to identify the genes that they regulate and thereby understand the role that these POU proteins play in sensory neuron gene regulation. We thank Action Research, the Cancer Research Campaign, the Sir Jules Thorn Trust and the Medical Research Council for supporting this work. 1. Latchman, 1). S. (1990) Gene Regulation: A Eukaryotic Perspective, Unwin Hyman, 1,ondon 2. Latchman, L). S. (1991) Eukaryotic Transcription Factors. Academic Press, I,ondon/San Diego 3. Johnson, 1’. I;. & McKnight, S. 1,. (19x0) Annu. Rev. Hiochem. 58,799-839 4. Mitchell, 1’. J. & Tjian, K. (19x9) Science 245, 371-378 5. Herr, W., Sturm, K. A,, Clerc, K.G., Corcoran, 1,. M., Baltimore, L)., Sharp, 1’. A., Ingraham, H. A,, Kosenfeld, M. G., Finney, M., Kuvkun, (;. 8( llorvitz. tI. K. (198X) Genes Lkv. 2, 15 13- 15 10 6. Ruvkun, G. & Finney, M. (1991) Cell 64,475-482 7. Ingraham, H. A., Chen, K., Mangalam, 11. J., I’lsholtz, H. P., Flynn, S. E., Linn, C. K.,Simmons, I). M., Swanson, 1,. & Kosenfeld, M. G. ( I O X X ) Cell 55, 519-529 8. Kristie, T. M. & Sharp, 1’. A. (1000) Genes Lkv. 4, 2383-2306 9. Scott, M. I)., Tamkun, J. W. & Hartzell, G.W. (IOXO) Hiochim. Iliophys. Acta 989. 25-48 10. Desai, C., Garrya, G., McIntire, S. I,. 8( IIorvitz, H. K. (19x8) Nature (London) 336, 038-h40 11. Johnson, W. A. & Hirsh, J. (1090) Nature (1,ondon) 343, 467-470 12. He, X., Treacy, M. N., Simmons, I). M., Ingraham, H. A,, Swanson, 1,. S. & Kosenfeld, M. G. (10x9) Nature (1.ondon) 340, 35-42 13. Wood, J. N., Hevan, S. J.. Coote, I’.. Ilarn, 1’. M., Hogan, I’., Latchman, I). S., Morrison. C.. Kougon. G., Theveniau, M. & Wheatley, S. C. ( I O O O ) I’roc. K. Soc. London I3 24 1, 187- 194 14. Suburo, A. M., Wheatley, S. C., Ilorn. I). A,. (”b son, S. J., Jahn, K., Fischer-Colbrie, K., Wood, J. N., Latchman, L). S. & I’olak, J. M. (1002) Neuroscience 46, XX 1-999 15. Sturm. K. A., L)as, G. & Herr, W. (10x3) Genes I k v . 2, 1582- 1590 16. Goldsborough. A,, Ashworth, A. & Willison, K. (1990) Nucleic Acids Kes. 18, 1634 17. IAycrop, K. A. & Latchman, L). S. (1091) Nucleic Acids Kes. 19, 3744 18. Clerc, K. G., Corcoran, I,. M., I,e Howitz, J. H., Baltimore, L). & Sharp, 1’. A. (19x8) Genes Lkv. 2, 1570- 158 1 19. IAlycrop, K. A.. Dent. C. I,., Wheatley, S. C.. Beech, M. N.. Ninkina, N. N., Wood, J. N. & Latchman. L). S. (1991) Neuron 7,381-390 20. Scheidereit, C., Heguy, k & Koeder. K. G. (1987) Cell 51. 783-793

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21. Scholar, 11. K.. I latzopoulos, A. K., Balling, K., Suzuki. N. & Grus. 1’. (1089) EMHO J. 8,2543-2550 22. I k n t , C. I,.. 1,illycrop. K. A,, Estridge. J., Thomas, N. S. H. & Latchman. I). S. (1991) Mol. Cell. Biol. 11. 302 5 - 39 30 23. Singh. H.. Sen. K., Haltimore, I). & Sharp, 1’. A. (1086) Nature (Imndon) 319. 151-158 24. Sive. II. I,.. fleintz, N. & Koeder. K. G. (1986) Mol. Cell. Iliol. 6, 3320-3340 2.S. I’ruijn, G. J. M., van 1)riei. W. & van der Vliet (1986) Nature (Idondon)322. hSh-050

20. Wood, J. N., I,illycrop, K. A,. 1)ent. C. I,., Minura. N. N., Beech, M. M.. Willoughby, J. J.. Winter, J. & Latchman. L). S. (1992)J. Biol. Chem., in press 27. Harade. Y.-A. (1989) Neuron 2, 1525- 1534 28. Wilcox. C. I,. & Johnson, E. M. (1987) J. Virol. 61,

2311-2315 20. Cushing, H. (1005) J. Am. Med. Assoc. 44. 1002- 1008 Keceived 16 April 1902

Comparisons of neuronal (PGP 9.5) and non-neuronal ubiquitin C-terminal hydrolases Keith D. Wilkinson, Seema Deshpande and Christopher N. Larsen Department of Biochemistry,Emory University School of Medicine, Atlanta, GA 30322, U.S.A.

Introduction lrbiquitin is a highly conserved eukaryotic protein that has been suggested to play a role in intracellular protein degradation [ 1 1, cell cycle regulation [2-41, DNA repair [ 5 1, recombination [6], chromatin structure [7], stress response [8, 91, programmed cell death [ 101, the mechanism of receptor action 11 1, 121 and ribosome biogenesis [ 13, 141. In all cases it appears to exert its function by being covalently attached to target proteins by an amide bond between the C-terminal glycine of ubiquitin and an a- or &-aminogroup on the attached protein [ 1, 151. At least three types of ubiquitin-protein bonds have been characterized. In the first type of linkage, ubiquitin-protein conjugates are linked by an amide bond between the C-terminus of ubiquitin and &-aminogroups on cellular proteins. This widespread post-translational modification of proteins is thought to target the attached protein for various metabolic fates [ 151. In the case of targeting for proteolysis (Fig. l), polyubiquitination of substrate proteins is observed. In this second type of linkage, poly-ubiquitin ‘chains’ are linked by amide bonds between the Cterminus of ubiquitin and the side chain amino group of I,ys-48 on the neighbouring ubiquitin. These poly-ubiquitinated proteins are recognized and degraded by specific proteases of the cell [ 11. 1 lydrolysis of this poly-ubiquitin chain must occur in order for ubiquitin to become available for another catalytic cycle. In contrast, the reversible ubiquitination of histones is limited to one or two

Abbreviations used: hsp, heat-shock protein; UCI-1, ubiquitin C-terminal hydrolase.

ubiquitin molecules per histone, does not lead to degradation, and may play a role in chromatin condensation or cell-cycle progression [2-4, 71. Covalent attachment of ubiquitin to the T-cell homing receptor [ 11 ] and the platelet-derived growth factor receptor [ 121 has been demonstrated but the consequences of this modification are unknown. Thus, proteins with covalently attached ubiquitin are present in the nucleus, the cytoplasm and the membranes of eukaryotic cells. In the third type of linkage, ubiquitin is linked to itself or other proteins via an a-amide linkage. Three eukaryotic genes [ 161 code for two proteins that consist of an N-terminal ubiquitin and a Cterminal ribosomal protein (Ub-CEP52 and UbCEPHO). Deletion of these genes results in cells that are defective in assembling intact ribosomes. It has recently been suggested that the expression of these ribosomal proteins as ubiquitin fusions may assist in ribosomal assembly but is not necessary for function [ 131. The ubiquitin is proteolytically processed from these proteins at a poorly defined point in the maturation of the ribosome. A fourth ubiquitin gene codes for a ubiquitin precursor (pro-IJb) containing 6- 12 repeats of the ubiquitin sequence. These repeats are arranged in a head-to-tail fashion with no linker peptides at the monomer junctions. A consensus heat-shock promoter is present in the 5’ regulatory region of this gene and ubiquitin is a heat-shock protein 18,91. Its role in stress response is undefined, but it may be induced to furnish enough new ubiquitin to accomplish the increased protein degradation observed in stress. Deletions of this gene in yeast result in cells that fail to sporulate, don’t survive heat-shock, and grow poorly under stress [17]. This pro-protein is very rapidly pro-

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POU family transcription factors in sensory neurons.

Nervous System-Specific Proteins POU family transcription factors in sensory neurons D. S. Latchman, C. L. Dent, K. A. Lillycrop and j. N. Wood* Medi...
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