- Email: [email protected]
The two activation domains of the Brn-3a transcription factor have distinct functional properties
Pergamon PII: Sl357-2725(97)00089-7
1357.171YY7
517
no + non
The Two Activation Domains of the Bm-3a Transcription Factor have Distinct Functional Properties JO L. BEGBIE,
DAVID
S. LATCHMAN”
Depurtment of Molecular Pathology, University College London Mrdicul School, The) Winde,\w Building, 46 Cleveland Street, London WlP 6DB, U.K. The Bm3a transcription factor has two distinct activation domains located, respectively, at the N-terminus and the C-terminus (coincident with the DNA binding POU domain) which differ in their effects on different target promoters. To analyze whether these regions could function when linked to a heterologous DNA binding domain, they were each linked to the GAL4 DNA binding domain. Here we show that the N-terminal domain constitutes a discrete activation domain which can function when linked to a heterologous DNA binding domain. Under these conditions this domain can activate at different distances upstream of the transcriptional start site hut does not do so when bound downstream of the start site. In contrast, the POC activation domain cannot activate transcription when delivered to DNA via a heterologous DNA binding domain, hut can function when linked to such a domain if hound to DNA via its own appropriate binding site. The reasons for this difference between these two domains are discussed in terms of the ability of the POU domain to bind to single stranded DNA and serve as a site for protein-protein interactions 0 1997 Elsevier Science Ltd. All rights reserved Keywords:
POU family transcription
factors
Activation
domains
Brn-3a
Int. J. Biochem. Cell Bid. (1997) 29. 1493-1500
INTRODUCTION
The Brn-3a transcription factor is a member of the POU (Pit-Ott-Uric) family of transcription factors originally identified on the basis of a common domain present in the mammalian transcription factors Pit- 1, Ott- 1 and Ott-2 and the nematode regulatory protein Uric-86 (for review, see Verrijzer and van der Vliet, 1993; Wegner et al., 1993). This common POU domain constitutes the DNA binding domain of these proteins and is composed of a POU-specific domain and a POU homeodomain which is related to the classical homeodomain found in other transcription factors (Gehring, 1987). *To whom all correspondence should Received 27 February 1997; accepted
be addressed. 16 June 1997. 1493
Brn-3a was originally isolated in a screen for novel POU factors expressed in the brain and was named Brn-3 (He ct al., 1989). Subsequently, however, it became clear that three related Brn-3 factors exist which are encoded by distinct genes (Theil et cd., 1994) and are expressed in distinct but overlapping patterns in the developing and adult nervous systems (Fedstova and Turner, 1996; Gerrero et al., 1993; Lillycrop et ul., 1992: Ninkina et al., 1993; Turner et al., 1994). The original Brn-3 factor was therefore renamed Brn-3a (also known as Brn-3.0; Gerrero et al., 1993; Lillycrop et al., 1992) whilst the other factors were named Brn-3b (also known as Brn-3.2; Lillycrop ez al., 1992; Turner et al., 1994) and Brn-3c (also known as Brn-3.1; Gerrero et nl., 1993; Ninkina et al., 1993). Of the mammalian POU factors so far identified, the Brn-3 fac-
1494
Jo L. Begbie
and David
tors show the closest homology to the Uric-86 factor whose inactivation leads to defects in neuronal development in the nematode (Desai et al., 1988; Finney et al., 1988). Hence, the Brn-3 factors are likely to play a critical role in the development of the mammalian nervous system. Indeed the inactivation of the Brn-3b gene in ‘knock out’ mice results in the absence of retinal neurons, whilst the similar inactivation of the Brn-3c gene leads to the absence of olfactory neurons (Erkman et al., 1996) and inactivation of Brn-3a leads to losses of both sensory and motor neurons (McEvilly et al., 1996). The apparently different roles of the different Brn-3 factors in viva is paralleled by differences in their functional effects on specific target genes. Thus, in transfection experiments, Brn-3a and Brn-3c activate a number of different promoters derived from neuronally expressed genes such as those encoding proopiomelanocortin (Gerrero et al., 1993), c(internexin (Budhram-Mahadeo et al., 1995) and SNAP-25 (Lakin et al., 1995), as well as an artificial promoter bearing their binding site upstream of the thymidine kinase (tk) promoter (Morris et al., 1994). In contrast, the basal activity of these promoters are inhibited by Brn-3b which also represses their activation by Brn-3a (Morris et al., 1994; BudhramMahadeo et a/., 1995; Morris et al., 1997). In experiments using chimaeric factors containing different regions derived from Brn-3a or Brn-3b, we have shown that the ability of Brn-3a to activate specific genes is dependent upon two distinct activation domains which differ in their ability to activate promoters (Budhram-Mahadeo et al., 1995, 1996; Morris et al., 1994). Thus, activation of the artificial promoter bearing Brn-3a binding sites linked to the tk promoter is dependent upon an activation domain located at the C-terminus coincident with the DNA binding POU domain and activation of this promoter can be achieved by the isolated Brn-3a POU domain (Budhram-Mahadeo et al., 1995, 1996; Morris et al., 1994). In contrast, activation of the c(internexin promoter cannot be achieved by the Brn-3a POU domain but is dependent upon a second activation domain located at the N-terminus of the molecule (Budhram-Mahadeo et
S. Latchnan
yeast GAL4 transcription factor and tested their effect on various promoters containing binding sites for this factor.
MATERIALS
AND
METHODS
Plasmid DNAs
The POU domain of Brn-3a was removed from the pJ4 vector (Morgenstein and Land, 1990) in which it was previously cloned (Budhram-Mahadeo et al., 1995) and subcloned into the pSGS424 vector downstream of the GAL4 DNA binding domain contained in this vector (Sadowski and Ptashne, 1987). The N-terminal region of Brn-3a containing regions I and II of the protein (amino acids l108; Budhram-Mahadeo et al., 1995; Morris et al., 1994) was isolated by PCR amplification of a full length Brn-3a cDNA and sub-cloned into the pGEM-T vector and then into pSGS424. All PCR products were sequenced to confirm their identity. The GST reporter construct containing two GAL4 sites at -94 relative to the transcription start site of the glutathione-S-transferase rr gene promoter (Cowell and Hurst, 1994) and the tk reporter constructs containing five GAL5 sites at different positions relative to the thymidine kinase promoter (Madden et ul., 1993). have previously been described. DNA trunsfection
Transfection of DNA was carried out according to the method of Gorman (Gorman, 1985). Routinely 1 x lo6 BHK-21 (Macpherson and Stoker, 1962) or ND7 cells (Wood et al., 1990) were transfected with 10 pg of the reporter plasmid and 10 pg of the expression vectors. In all cases, cells were harvested 72 h later. The amount of DNA taken up by the cells in each case was measured by slot blotting the extract and hybridization with a probe derived from the ampicillin resistance gene in the plasmid vector (Abken and Reifenrath, 1992). This value was then used to normalize the values obtained in the chloramphenicol acetyltransferase assay as a control for differences in uptake of plasmid DNA in each sample.
al., 1995).
Choramphenicol
acetyltransferase assay
To investigate these effects further, we have linked each of the two activation domains of Brn-3a to the DNA binding domain of the
Assays of chloramphenicol acetyltransferase activity were carried out according to the method of Gorman (Gorman, 1985) using
Bm-3a
transcription
facto1
samples that had been equalized for protein content as described by the method of Bradford, (1976). The values for CAT activity obtained in this way were then adjusted on the basis of the values obtained in the plasmid uptake assay as described above. RESULTS
AND
DISCUSSION
The N-terminal or POU region activation domains of Brn-3a were linked to the DNA binding domain of GAL4 in the mammalian expression vector pSGS424 (Sadowski and Ptashne, 1989). Similar constructs were also prepared expressing the POU domain of either Brn-3b or Brn-3c Zfor comparison. Each of these constructs were then transfected into BHK-21 fibroblast cells (Macpheson and Stoker, 1962) which lack endogenous Brn-3 (Lillycrop et al., 1992) together with the glutathione-S-transferase 7c gene promoter containing two GAL4 DNA binding sites cloned at -94 bases upstream of the gene encoding the readily assayable chloramphenicol acetyl transferase (CAT) protein (Cowell and Hurst, 1994). The degree of CAT activity observed in each case was compared to that obtained upon transfection of the target promoter with the pSGS424 vector lacking any insert. In these experiments (Fig. la) the N-terminal domain of Brn-3a was able to activate the promoter approximately 40-fold indicating that it functioned as a strong activation domain under these conditions. In contrast, the POU domain of Brn-3a linked to GAL4 had no effect on the promoter and a similar lack of effect was also observed for the POU domains of Brn-3b and Brn-3c. Strong activation of the promoter by the N-terminal domain but not by the POU domain was also observed upon co-transfection of ND7 neuronal cells which express endogenous Brn-3s (Lillycrop et al., 1992) indicating that these effects are not dependent upon the presence or absence of endogenous Brn-3 (Fig. lb). To further investigate the activity of the Nterminal domain, we utilized a series of constructs (Madden et al., 1993) which contained five GAL4 binding sites cloned at various positions relative to the transcriptional start site of the thymidine kinase (tk) promoter. As shown in Fig. 2a, the N-terminal activation domain was able to activate a tk promoter containing the GAL4 sites at -105 bases upstream of the transcriptional start site paral-
Construct
I750
c
(b)
Construct Fig. 1. Cat assay results showing the effect of transfecting either BHK-21 fibroblast cells (panel a) or ND7 neuronal cells (panel b) with the GST-GAL4 -Cat construct and either pSGS424 vector expressing only the DNA binding domain of GAL4, or the same vector containing the indicated Brn-3 region fused to the DNA binding domain of GAL4. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 1009/o). Values are the average of three determinations whose ytandard error is shown by the bars.
leling their position in the GST promoter, although the magnitude of the response was less in the case of the tk promoter. In contrast the POU domain of Brn-3a if anything had an inhibitory effect on the GAL4/tk promoter construct. Hence the effects we observed with the GST promoter are not promoter specific
I496
Jo L. Begbte
and David
z Construct
700
r
(b)
3 400 e, z2 300 8 aE 200 100
Fig. 2. Cat assay showing the results of co-transfecting BHK cells with constructs containing the indicated region of Brn-3 cloned into the pSGS424 vector and constructs containing the tk promoter with GAL4 sites at -105 (panel a), -760 (panel b), or + 1000 (panel c), relative to
S. Latchman
but can also be observed with a different promoter. Interestingly the N-terminal domain was also able to strongly stimulate a tk promoter construct in which the GAL4 sites were positioned further upstream relative to the transcriptional start site (Fig. 2b) but had no stimulatory effect when the GAL4 sites were positioned downstream of the transcriptional start site (Fig. 2~). As before, the Brn-3a POU domain construct had an inhibitory effect on the reporter constructs. The N-terminal domain of Brn-3a thus represents a discrete activation domain capable of activating transcription, although its activity is clearly affected by its position (upstream or downstream) relative to the transcriptional start site. It is possible, therefore, that this activation domain may need to be in a particular orientation relative to the basal transcriptional complex in order to stimulate its activity, thereby rendering it inactive when bound downstream of the transcriptional start site. Interestingly, the N-terminal region of Brn3a is subject to alternative splicing to produce long and short forms of the protein and this process is regulated during neuronal differentiation (Liu et al., 1996). The short form of the protein lacks the 84 most N-terminal amino acids present in the long form of the protein and hence contains only a part of the N-terminal domain (amino acids l&108) which is fused to GAL4 in our construct. This results in the short form, unlike the long form, being unable to co-operate with the ras oncogene to transform primary fibroblasts (Theil et al., 1993) and prevents it activating some promoters which are activated by the long form of Brn-3a (Budhram-Mahadeo et al., 1995). To investigate the contribution to the activity of the activation domain made by the region of it which is unique to the long form of Brn-3a, we prepared a construct in which this unique region was linked to GAL4. When tested on the two different promoters, this region was able to stimulate both the GST promoter (Fig. 3a) and the tk promoter (Fig. 3b) but its effect was about 223-fold less than that observed with the full N-terminal the transcriptional start site. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%). Values are the average of three determinations whose standard error is shown by the bars.
Bm-3a
transcription
Fig. 3. Cat assay showing the results of transfecting BHK cells with either the GST (panel a) or -105 tk (panel b) reporter constructs containing either the Brn-3a POU domain. the entire N-terminal region used in previous experiments (N-term I and s). or the extreme N-terminal region unique to the long form of Bm-3a (N-term 1). In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%).
region. Hence, the extreme N-terminal region unique to the long form of Brn-3a can stimulate gene expression but the adjacent region common to both forms is required for maximal activity. Although the N-terminal domain of Brn-3a is, therefore, a conventional activation domain capable of stimulating gene expression when linked to GAL4, the POU domain of Brn-3a was unable to stimulate promoter activity in these assays (see Figs 1 and 2). To confirm that this effect was not due to some defect in
factor
I497
the construct, we co-transfected the G4L4: Brn-3a POU domain vector with a reporter construct containing an octamer binding site for Brn-3a instead of the GAL4 sites upstream of the tk promoter. As show in Fig. 4a. this reporter construct was indeed activated by the GAL4/Brn-3a POU domain construct. In contrast the Brn-3b and Brn-3c POU domain constructs had no effect on this promoter and a similar lack of effect was observed. as expected, with the GAL4/N-terminal domain construct which would be unable to bind to a promoter lacking GAL4 sites. Although the stimulatory effect ol‘ the GAL4/Brn-3a POU domain construct on this promoter was relatively small, it was comparable to that observed on the same promoter with a construct containing only the Brn-3a POU domain without the GAL4 region (Fig. 4b) used in our previous studies (Budhram-Mahadeo et ul., 1995, 1996). Interestingly, the Brn-3c POU domain which we had not previously tested had no effect on the promoter either alone or linked to GAL4 (Fig. 4). Thus. the ability of full length Rrn-3c to stimulate specific promoters (Morris or (I/., 1994) must be dependent on a region outside the POU domain. In the case of Brn-3a, it is clear that the POU domain can only function as an activation domain when it directs DNA binding to its appropriate target sequence and not when it is brought to the DNA via another DNA binding domain. A similar finding has also been reported for the serum response factor which when linked to the DNA binding domain of the bacterial LexA factor, produces strong activation when bound to its target serum response element, but only weah activation when bound to a LexA binding site (Hill et ul., 1994). In the case of Brn-3a, we have previously shown that the ability of the POU domain to activate transcription is dependent on the context of its target binding site (BudhramMahadeo et rrl., 1996). Thus. although the POU domain cannot activate the z-internexin promoter (Budhram-Mahadeo et cl/., 1995). it can do so when the Brn-3a target site in the xinternexin promoter is moved to a ditl’erent context within the tk promoter (BudhramMahadeo et al., 1996). When taken together with the ability of Brn-3a and Brn-3b to bind to single stranded DNA (Budhram-Mahadeo et ul.. 1996). this suggests the possibility that
Jo L. Begbie
d
a
a
c 5 z
and David
>
construct
Fig. 4. Cat assay showing the result of transfecting BHK21 cells with constructs containing the indicated regions of Brn-3 either linked to the DNA binding domain of GAL4 (panel a), or expressed in isolation in the pJ4 vector (panel b) and a tk reporter construct containing octamer binding sites for Brn-3a at -105 relative to the transcriptional start site. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%).
the binding of Brn-3a may alter the structure of appropriate adjacent DNA sequences, thus allowing other activators to bind and stimulate transcription. No such changes would evidently occur when DNA binding takes place via the GAL4 DNA binding domain preventing transcriptional activation from occuring. Alternatively, it is possible that, when bound to its appropriate binding site, the Brn-
S. Latchman
3a POU domain undergoes a conformational change which allows it to be recognized by a co-activating molecule necessary for transcriptional activation. Thus, in the case of the related POU factor Ott-1, binding to a TAATGARAT (R = purine) element results in a conformational change which allows Oct1 to be recognized by the herpes simplex virus virion protein Vmw65 (Walker et al., 1994). This change does not occur when Ott-I is bound to a conventional octamer motif preventing Vmw65 from binding. Thus, Vmw65 activates transcription only via a TAATGARAT sequence and not via a conventional motif. Interestingly, the amino acid at position 22 in the POU-homeodomain appears to play a critical role both in the interaction of Ott-1 with Vmw65 and the ability of the Brn-3a POU domain to activate transcription. Thus, exchange of the alanine found at this position in Ott-2 for the glutamic acid found in Ott-1 allows Ott-2 to interact with Vmw65 when it can not normally do so. Similarly, replacement of the valine found at this position in the POU homeodomain of Brn-3a with the isoleutine found at the equivalent position in Brn3b abolishes its ability to activate transcription, while the converse substitution allows Brn-3b to activate transcription (Dawson et al., 1996). Hence, this site is likely to serve as a critical site for protein-protein interactions of POU proteins with co-activating molecules, which, at least in the case of Ott-1, require a conformational change induced by DNA binding to an appropriate binding site. If this is the case for Brn-3a also, this change in protein configuration would evidently not occur on binding to a GAL4 binding site preventing transcriptional activation from occuring. Although further experiments will be required to investigate this possibility, it is already clear that the two activation domains of Brn-3a have distinct properties, with the Nterminal domain acting as a transferable domain which can activate gene expression when linked to a heterologous DNA binding domain, whereas the POU domain can only activate transcription when it has directed DNA binding to an appropriate binding site.
Acknowledgements--We thank cussions. JLB was supported Council CASE studentship.
John Wood for helpful disby a Medical Research
Brn-3a
transcl ription
REFERENCES Abken H. and Reifenrath B. (1992) A procedure to standardize CAT reporter gene assay. Nucl. Acids Res. 20, 3527. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. A&. Biochrm 72, 248 -254. Budhram-Mahadeo V., Morris P. J., Lakin N. D., Theil T.. Ching G. Y.. Lillycrop K. A., Moroy T., Liem R. K. H. and Latchman D. S. (1995) Activation of the xinternexin promoter by the Brn-3a transcription factor is dependent on the N-terminal region of the protein. J. Biol. Chew. 270, 2853 ~2858. Budhram-Mahadeo V., Morris P. J., Lakin N. D., Dawson S. J. and Latchman D. S. (1996) The different activities of the two activation domains of the Brn-3a transcription factor are dependent on the context of the binding site. .1. Biol. Chem. 271, 910X-91 13. Cowell 1. Ci. and Hurst H. C. (1994) Transcriptional repression by the human b Zip factor E4 BP4: definition of a minimal repression domain. Nucl. Acids Rcs. 22, 59 65. Dawson S. J., Morris P. J. and Latchman D. S. (1996) A single amino acid change converts an inhibiting transcription factor into an activator. J. Biol. Chem. 271, II631 11633. Desal c‘., Garriga G.. McIntire S. L. and Horvitz H. R. (1988) A genetic pathway for the development of Caenorhabditis elegans HSN motor neurons. Nature 336, 63X 646. Erkman L.. McEvilly J.. Luo L., Ryan A. K., Hooshmand F.. O’Connel S. M.. Keithley E. M., Rapaport D. H., Ryan A. F. and Rosenfeld M. G. (1996) Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381, 603-606. Fedthova N. G. and Turner E. E. (1996) Brn-3.0 exprcssion identifies early pot-mitotic CNS neurons and sensory neural precursors. Mech. Dev. 53, 291-304. Finncy M., Ruvkin Cr. and Horvitz H. R. (1988) The C. e/c,,gtrn.\ cell lineage and differentiation gene uric-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell 55, 757--769. Gehring W. J. (I 987) Homeoboxes in the study of development. &ienw 236, 1245 -1252. Gerrero M. R.. McEvilly R. J., Tuner E., Lin C. R., O’Connell S., Jenne K. J., Hobbs M. V. and Rosenfeld M. G. ( 1993) Brn-3.0: a POU domain protein expressed in the sensory immune and endocrine systems that functions on elements distinct from known octamer motifs. Ptw. Narl. Awl. Sci., U.S.A. 90, 10841-10845. German C. M. (1985) High efficiency gene transfer into mammalian cells. In DNA cloning. a practical approuch. ed. D. M. Clover, pp. 143~-190 (IRL Press Oxford). He X.. Treaty M. N.. Simmons D. M., lngraham H. A., Swmsno L. S. and Rosenfeld M. G. (1989) Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340, 35-42. Hill C. S., Wynne J. and Treisman R. (1994) Serum-regulated transcription by serum response factor (SRF): a novel role for the DNA binding domain. EMBO J. 13, 5421 5432. Lakin N. D.. Morris P. J.. Theil T., Sato T. N.. Moroy T.. Wilson M. C. and Latchman D. S. (1995) Regulation of neurite outgrowth and SNAP-25 gene ex-
factor
I199
pression by the Brn-3a transcription factor. .I Viol. Chem. 270, 15858-15X63. Lillycrop K. A., Budhram-Mahadeo V. S., Lakin N. D., Terrenghi G.. Wood J. N., Polak J. M. and Latchman D. S. (1992) A novel POU family transcription factor is closely related to Brn-3 but has a distinct expression pattern in neuronal cells. .vuc,/. A&Y Rcsv. 20, 5093 5096. Liu Y. Z.. Dawson S. J. and Latchman I>. S. i 14Q6) Alternative splicing of the Brn-3a and Brn-3b transcrlption factor mRNAs is regulated in neuronal cells ./. Mol. Neurosci. 7, 77.~85. McEvilly R. J.. Erkman L., Luo L.. Sawchenko P. E.. Ryan A. F. and Rosenfeld M. G. (1996) Requirement for Brn-3.0 in differentiation and survival of \cnsory and motor neurons. Nature 384, 574 577. Macpherson I. and Stoker M. (1962) Polyma tranxl’ornation of hamster cell clones- -an investigation 01‘ the genetic factors affecting cell competence. [‘irol~rr,’ 16, 147~151. Madden S. L.. Cook D. M. and Rauscher F. J. (1993) A structure-function analysis of transcriptional repression mediated by the WTI Wilms’ tumour suppressor protein. Onccqyw 8, 17 I3- 1720. Morgenstern J. P. and Land H. (1990) A series of mammalian expression vectors and Characterization 01’ their expression of a reporter gene in stably and tran\sently transfected cells. Ni,c/. Acids Rex. 18, 1068. Morris P. J.. Theil T., Ring C. J. A.. Lillycrop K. A,Miir
I500
Jo L. Begbie
and David
Wegner M.. Drolet D. W. and Rosenfeld M. G. (1993) POU-domain proteins: structure and function of developmental regulators. Curr. Opin. Cell Biol. 5, 488-498. Wood J. N., Bevan S. J., Coote P.. Darn P. M., Hogan P., Latchman D. S., Morrison C., Rougon G..
S. Latchman Theveniau M. and Wheatley S. C. (1990) Novel cell lines display the properties of nociceptive sensory neurons. Proc. Rovul Sot. London Serks B 241, 187 194.
1357.171YY7
517
no + non
The Two Activation Domains of the Bm-3a Transcription Factor have Distinct Functional Properties JO L. BEGBIE,
DAVID
S. LATCHMAN”
Depurtment of Molecular Pathology, University College London Mrdicul School, The) Winde,\w Building, 46 Cleveland Street, London WlP 6DB, U.K. The Bm3a transcription factor has two distinct activation domains located, respectively, at the N-terminus and the C-terminus (coincident with the DNA binding POU domain) which differ in their effects on different target promoters. To analyze whether these regions could function when linked to a heterologous DNA binding domain, they were each linked to the GAL4 DNA binding domain. Here we show that the N-terminal domain constitutes a discrete activation domain which can function when linked to a heterologous DNA binding domain. Under these conditions this domain can activate at different distances upstream of the transcriptional start site hut does not do so when bound downstream of the start site. In contrast, the POC activation domain cannot activate transcription when delivered to DNA via a heterologous DNA binding domain, hut can function when linked to such a domain if hound to DNA via its own appropriate binding site. The reasons for this difference between these two domains are discussed in terms of the ability of the POU domain to bind to single stranded DNA and serve as a site for protein-protein interactions 0 1997 Elsevier Science Ltd. All rights reserved Keywords:
POU family transcription
factors
Activation
domains
Brn-3a
Int. J. Biochem. Cell Bid. (1997) 29. 1493-1500
INTRODUCTION
The Brn-3a transcription factor is a member of the POU (Pit-Ott-Uric) family of transcription factors originally identified on the basis of a common domain present in the mammalian transcription factors Pit- 1, Ott- 1 and Ott-2 and the nematode regulatory protein Uric-86 (for review, see Verrijzer and van der Vliet, 1993; Wegner et al., 1993). This common POU domain constitutes the DNA binding domain of these proteins and is composed of a POU-specific domain and a POU homeodomain which is related to the classical homeodomain found in other transcription factors (Gehring, 1987). *To whom all correspondence should Received 27 February 1997; accepted
be addressed. 16 June 1997. 1493
Brn-3a was originally isolated in a screen for novel POU factors expressed in the brain and was named Brn-3 (He ct al., 1989). Subsequently, however, it became clear that three related Brn-3 factors exist which are encoded by distinct genes (Theil et cd., 1994) and are expressed in distinct but overlapping patterns in the developing and adult nervous systems (Fedstova and Turner, 1996; Gerrero et al., 1993; Lillycrop et ul., 1992: Ninkina et al., 1993; Turner et al., 1994). The original Brn-3 factor was therefore renamed Brn-3a (also known as Brn-3.0; Gerrero et al., 1993; Lillycrop et al., 1992) whilst the other factors were named Brn-3b (also known as Brn-3.2; Lillycrop ez al., 1992; Turner et al., 1994) and Brn-3c (also known as Brn-3.1; Gerrero et nl., 1993; Ninkina et al., 1993). Of the mammalian POU factors so far identified, the Brn-3 fac-
1494
Jo L. Begbie
and David
tors show the closest homology to the Uric-86 factor whose inactivation leads to defects in neuronal development in the nematode (Desai et al., 1988; Finney et al., 1988). Hence, the Brn-3 factors are likely to play a critical role in the development of the mammalian nervous system. Indeed the inactivation of the Brn-3b gene in ‘knock out’ mice results in the absence of retinal neurons, whilst the similar inactivation of the Brn-3c gene leads to the absence of olfactory neurons (Erkman et al., 1996) and inactivation of Brn-3a leads to losses of both sensory and motor neurons (McEvilly et al., 1996). The apparently different roles of the different Brn-3 factors in viva is paralleled by differences in their functional effects on specific target genes. Thus, in transfection experiments, Brn-3a and Brn-3c activate a number of different promoters derived from neuronally expressed genes such as those encoding proopiomelanocortin (Gerrero et al., 1993), c(internexin (Budhram-Mahadeo et al., 1995) and SNAP-25 (Lakin et al., 1995), as well as an artificial promoter bearing their binding site upstream of the thymidine kinase (tk) promoter (Morris et al., 1994). In contrast, the basal activity of these promoters are inhibited by Brn-3b which also represses their activation by Brn-3a (Morris et al., 1994; BudhramMahadeo et a/., 1995; Morris et al., 1997). In experiments using chimaeric factors containing different regions derived from Brn-3a or Brn-3b, we have shown that the ability of Brn-3a to activate specific genes is dependent upon two distinct activation domains which differ in their ability to activate promoters (Budhram-Mahadeo et al., 1995, 1996; Morris et al., 1994). Thus, activation of the artificial promoter bearing Brn-3a binding sites linked to the tk promoter is dependent upon an activation domain located at the C-terminus coincident with the DNA binding POU domain and activation of this promoter can be achieved by the isolated Brn-3a POU domain (Budhram-Mahadeo et al., 1995, 1996; Morris et al., 1994). In contrast, activation of the c(internexin promoter cannot be achieved by the Brn-3a POU domain but is dependent upon a second activation domain located at the N-terminus of the molecule (Budhram-Mahadeo et
S. Latchnan
yeast GAL4 transcription factor and tested their effect on various promoters containing binding sites for this factor.
MATERIALS
AND
METHODS
Plasmid DNAs
The POU domain of Brn-3a was removed from the pJ4 vector (Morgenstein and Land, 1990) in which it was previously cloned (Budhram-Mahadeo et al., 1995) and subcloned into the pSGS424 vector downstream of the GAL4 DNA binding domain contained in this vector (Sadowski and Ptashne, 1987). The N-terminal region of Brn-3a containing regions I and II of the protein (amino acids l108; Budhram-Mahadeo et al., 1995; Morris et al., 1994) was isolated by PCR amplification of a full length Brn-3a cDNA and sub-cloned into the pGEM-T vector and then into pSGS424. All PCR products were sequenced to confirm their identity. The GST reporter construct containing two GAL4 sites at -94 relative to the transcription start site of the glutathione-S-transferase rr gene promoter (Cowell and Hurst, 1994) and the tk reporter constructs containing five GAL5 sites at different positions relative to the thymidine kinase promoter (Madden et ul., 1993). have previously been described. DNA trunsfection
Transfection of DNA was carried out according to the method of Gorman (Gorman, 1985). Routinely 1 x lo6 BHK-21 (Macpherson and Stoker, 1962) or ND7 cells (Wood et al., 1990) were transfected with 10 pg of the reporter plasmid and 10 pg of the expression vectors. In all cases, cells were harvested 72 h later. The amount of DNA taken up by the cells in each case was measured by slot blotting the extract and hybridization with a probe derived from the ampicillin resistance gene in the plasmid vector (Abken and Reifenrath, 1992). This value was then used to normalize the values obtained in the chloramphenicol acetyltransferase assay as a control for differences in uptake of plasmid DNA in each sample.
al., 1995).
Choramphenicol
acetyltransferase assay
To investigate these effects further, we have linked each of the two activation domains of Brn-3a to the DNA binding domain of the
Assays of chloramphenicol acetyltransferase activity were carried out according to the method of Gorman (Gorman, 1985) using
Bm-3a
transcription
facto1
samples that had been equalized for protein content as described by the method of Bradford, (1976). The values for CAT activity obtained in this way were then adjusted on the basis of the values obtained in the plasmid uptake assay as described above. RESULTS
AND
DISCUSSION
The N-terminal or POU region activation domains of Brn-3a were linked to the DNA binding domain of GAL4 in the mammalian expression vector pSGS424 (Sadowski and Ptashne, 1989). Similar constructs were also prepared expressing the POU domain of either Brn-3b or Brn-3c Zfor comparison. Each of these constructs were then transfected into BHK-21 fibroblast cells (Macpheson and Stoker, 1962) which lack endogenous Brn-3 (Lillycrop et al., 1992) together with the glutathione-S-transferase 7c gene promoter containing two GAL4 DNA binding sites cloned at -94 bases upstream of the gene encoding the readily assayable chloramphenicol acetyl transferase (CAT) protein (Cowell and Hurst, 1994). The degree of CAT activity observed in each case was compared to that obtained upon transfection of the target promoter with the pSGS424 vector lacking any insert. In these experiments (Fig. la) the N-terminal domain of Brn-3a was able to activate the promoter approximately 40-fold indicating that it functioned as a strong activation domain under these conditions. In contrast, the POU domain of Brn-3a linked to GAL4 had no effect on the promoter and a similar lack of effect was also observed for the POU domains of Brn-3b and Brn-3c. Strong activation of the promoter by the N-terminal domain but not by the POU domain was also observed upon co-transfection of ND7 neuronal cells which express endogenous Brn-3s (Lillycrop et al., 1992) indicating that these effects are not dependent upon the presence or absence of endogenous Brn-3 (Fig. lb). To further investigate the activity of the Nterminal domain, we utilized a series of constructs (Madden et al., 1993) which contained five GAL4 binding sites cloned at various positions relative to the transcriptional start site of the thymidine kinase (tk) promoter. As shown in Fig. 2a, the N-terminal activation domain was able to activate a tk promoter containing the GAL4 sites at -105 bases upstream of the transcriptional start site paral-
Construct
I750
c
(b)
Construct Fig. 1. Cat assay results showing the effect of transfecting either BHK-21 fibroblast cells (panel a) or ND7 neuronal cells (panel b) with the GST-GAL4 -Cat construct and either pSGS424 vector expressing only the DNA binding domain of GAL4, or the same vector containing the indicated Brn-3 region fused to the DNA binding domain of GAL4. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 1009/o). Values are the average of three determinations whose ytandard error is shown by the bars.
leling their position in the GST promoter, although the magnitude of the response was less in the case of the tk promoter. In contrast the POU domain of Brn-3a if anything had an inhibitory effect on the GAL4/tk promoter construct. Hence the effects we observed with the GST promoter are not promoter specific
I496
Jo L. Begbte
and David
z Construct
700
r
(b)
3 400 e, z2 300 8 aE 200 100
Fig. 2. Cat assay showing the results of co-transfecting BHK cells with constructs containing the indicated region of Brn-3 cloned into the pSGS424 vector and constructs containing the tk promoter with GAL4 sites at -105 (panel a), -760 (panel b), or + 1000 (panel c), relative to
S. Latchman
but can also be observed with a different promoter. Interestingly the N-terminal domain was also able to strongly stimulate a tk promoter construct in which the GAL4 sites were positioned further upstream relative to the transcriptional start site (Fig. 2b) but had no stimulatory effect when the GAL4 sites were positioned downstream of the transcriptional start site (Fig. 2~). As before, the Brn-3a POU domain construct had an inhibitory effect on the reporter constructs. The N-terminal domain of Brn-3a thus represents a discrete activation domain capable of activating transcription, although its activity is clearly affected by its position (upstream or downstream) relative to the transcriptional start site. It is possible, therefore, that this activation domain may need to be in a particular orientation relative to the basal transcriptional complex in order to stimulate its activity, thereby rendering it inactive when bound downstream of the transcriptional start site. Interestingly, the N-terminal region of Brn3a is subject to alternative splicing to produce long and short forms of the protein and this process is regulated during neuronal differentiation (Liu et al., 1996). The short form of the protein lacks the 84 most N-terminal amino acids present in the long form of the protein and hence contains only a part of the N-terminal domain (amino acids l&108) which is fused to GAL4 in our construct. This results in the short form, unlike the long form, being unable to co-operate with the ras oncogene to transform primary fibroblasts (Theil et al., 1993) and prevents it activating some promoters which are activated by the long form of Brn-3a (Budhram-Mahadeo et al., 1995). To investigate the contribution to the activity of the activation domain made by the region of it which is unique to the long form of Brn-3a, we prepared a construct in which this unique region was linked to GAL4. When tested on the two different promoters, this region was able to stimulate both the GST promoter (Fig. 3a) and the tk promoter (Fig. 3b) but its effect was about 223-fold less than that observed with the full N-terminal the transcriptional start site. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%). Values are the average of three determinations whose standard error is shown by the bars.
Bm-3a
transcription
Fig. 3. Cat assay showing the results of transfecting BHK cells with either the GST (panel a) or -105 tk (panel b) reporter constructs containing either the Brn-3a POU domain. the entire N-terminal region used in previous experiments (N-term I and s). or the extreme N-terminal region unique to the long form of Bm-3a (N-term 1). In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%).
region. Hence, the extreme N-terminal region unique to the long form of Brn-3a can stimulate gene expression but the adjacent region common to both forms is required for maximal activity. Although the N-terminal domain of Brn-3a is, therefore, a conventional activation domain capable of stimulating gene expression when linked to GAL4, the POU domain of Brn-3a was unable to stimulate promoter activity in these assays (see Figs 1 and 2). To confirm that this effect was not due to some defect in
factor
I497
the construct, we co-transfected the G4L4: Brn-3a POU domain vector with a reporter construct containing an octamer binding site for Brn-3a instead of the GAL4 sites upstream of the tk promoter. As show in Fig. 4a. this reporter construct was indeed activated by the GAL4/Brn-3a POU domain construct. In contrast the Brn-3b and Brn-3c POU domain constructs had no effect on this promoter and a similar lack of effect was observed. as expected, with the GAL4/N-terminal domain construct which would be unable to bind to a promoter lacking GAL4 sites. Although the stimulatory effect ol‘ the GAL4/Brn-3a POU domain construct on this promoter was relatively small, it was comparable to that observed on the same promoter with a construct containing only the Brn-3a POU domain without the GAL4 region (Fig. 4b) used in our previous studies (Budhram-Mahadeo et ul., 1995, 1996). Interestingly, the Brn-3c POU domain which we had not previously tested had no effect on the promoter either alone or linked to GAL4 (Fig. 4). Thus. the ability of full length Rrn-3c to stimulate specific promoters (Morris or (I/., 1994) must be dependent on a region outside the POU domain. In the case of Brn-3a, it is clear that the POU domain can only function as an activation domain when it directs DNA binding to its appropriate target sequence and not when it is brought to the DNA via another DNA binding domain. A similar finding has also been reported for the serum response factor which when linked to the DNA binding domain of the bacterial LexA factor, produces strong activation when bound to its target serum response element, but only weah activation when bound to a LexA binding site (Hill et ul., 1994). In the case of Brn-3a, we have previously shown that the ability of the POU domain to activate transcription is dependent on the context of its target binding site (BudhramMahadeo et rrl., 1996). Thus. although the POU domain cannot activate the z-internexin promoter (Budhram-Mahadeo et cl/., 1995). it can do so when the Brn-3a target site in the xinternexin promoter is moved to a ditl’erent context within the tk promoter (BudhramMahadeo et al., 1996). When taken together with the ability of Brn-3a and Brn-3b to bind to single stranded DNA (Budhram-Mahadeo et ul.. 1996). this suggests the possibility that
Jo L. Begbie
d
a
a
c 5 z
and David
>
construct
Fig. 4. Cat assay showing the result of transfecting BHK21 cells with constructs containing the indicated regions of Brn-3 either linked to the DNA binding domain of GAL4 (panel a), or expressed in isolation in the pJ4 vector (panel b) and a tk reporter construct containing octamer binding sites for Brn-3a at -105 relative to the transcriptional start site. In all cases, the Cat activity observed is compared to that observed upon co-transfection of the reporter construct with empty expression vector (set at 100%).
the binding of Brn-3a may alter the structure of appropriate adjacent DNA sequences, thus allowing other activators to bind and stimulate transcription. No such changes would evidently occur when DNA binding takes place via the GAL4 DNA binding domain preventing transcriptional activation from occuring. Alternatively, it is possible that, when bound to its appropriate binding site, the Brn-
S. Latchman
3a POU domain undergoes a conformational change which allows it to be recognized by a co-activating molecule necessary for transcriptional activation. Thus, in the case of the related POU factor Ott-1, binding to a TAATGARAT (R = purine) element results in a conformational change which allows Oct1 to be recognized by the herpes simplex virus virion protein Vmw65 (Walker et al., 1994). This change does not occur when Ott-I is bound to a conventional octamer motif preventing Vmw65 from binding. Thus, Vmw65 activates transcription only via a TAATGARAT sequence and not via a conventional motif. Interestingly, the amino acid at position 22 in the POU-homeodomain appears to play a critical role both in the interaction of Ott-1 with Vmw65 and the ability of the Brn-3a POU domain to activate transcription. Thus, exchange of the alanine found at this position in Ott-2 for the glutamic acid found in Ott-1 allows Ott-2 to interact with Vmw65 when it can not normally do so. Similarly, replacement of the valine found at this position in the POU homeodomain of Brn-3a with the isoleutine found at the equivalent position in Brn3b abolishes its ability to activate transcription, while the converse substitution allows Brn-3b to activate transcription (Dawson et al., 1996). Hence, this site is likely to serve as a critical site for protein-protein interactions of POU proteins with co-activating molecules, which, at least in the case of Ott-1, require a conformational change induced by DNA binding to an appropriate binding site. If this is the case for Brn-3a also, this change in protein configuration would evidently not occur on binding to a GAL4 binding site preventing transcriptional activation from occuring. Although further experiments will be required to investigate this possibility, it is already clear that the two activation domains of Brn-3a have distinct properties, with the Nterminal domain acting as a transferable domain which can activate gene expression when linked to a heterologous DNA binding domain, whereas the POU domain can only activate transcription when it has directed DNA binding to an appropriate binding site.
Acknowledgements--We thank cussions. JLB was supported Council CASE studentship.
John Wood for helpful disby a Medical Research
Brn-3a
transcl ription
REFERENCES Abken H. and Reifenrath B. (1992) A procedure to standardize CAT reporter gene assay. Nucl. Acids Res. 20, 3527. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. A&. Biochrm 72, 248 -254. Budhram-Mahadeo V., Morris P. J., Lakin N. D., Theil T.. Ching G. Y.. Lillycrop K. A., Moroy T., Liem R. K. H. and Latchman D. S. (1995) Activation of the xinternexin promoter by the Brn-3a transcription factor is dependent on the N-terminal region of the protein. J. Biol. Chew. 270, 2853 ~2858. Budhram-Mahadeo V., Morris P. J., Lakin N. D., Dawson S. J. and Latchman D. S. (1996) The different activities of the two activation domains of the Brn-3a transcription factor are dependent on the context of the binding site. .1. Biol. Chem. 271, 910X-91 13. Cowell 1. Ci. and Hurst H. C. (1994) Transcriptional repression by the human b Zip factor E4 BP4: definition of a minimal repression domain. Nucl. Acids Rcs. 22, 59 65. Dawson S. J., Morris P. J. and Latchman D. S. (1996) A single amino acid change converts an inhibiting transcription factor into an activator. J. Biol. Chem. 271, II631 11633. Desal c‘., Garriga G.. McIntire S. L. and Horvitz H. R. (1988) A genetic pathway for the development of Caenorhabditis elegans HSN motor neurons. Nature 336, 63X 646. Erkman L.. McEvilly J.. Luo L., Ryan A. K., Hooshmand F.. O’Connel S. M.. Keithley E. M., Rapaport D. H., Ryan A. F. and Rosenfeld M. G. (1996) Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381, 603-606. Fedthova N. G. and Turner E. E. (1996) Brn-3.0 exprcssion identifies early pot-mitotic CNS neurons and sensory neural precursors. Mech. Dev. 53, 291-304. Finncy M., Ruvkin Cr. and Horvitz H. R. (1988) The C. e/c,,gtrn.\ cell lineage and differentiation gene uric-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell 55, 757--769. Gehring W. J. (I 987) Homeoboxes in the study of development. &ienw 236, 1245 -1252. Gerrero M. R.. McEvilly R. J., Tuner E., Lin C. R., O’Connell S., Jenne K. J., Hobbs M. V. and Rosenfeld M. G. ( 1993) Brn-3.0: a POU domain protein expressed in the sensory immune and endocrine systems that functions on elements distinct from known octamer motifs. Ptw. Narl. Awl. Sci., U.S.A. 90, 10841-10845. German C. M. (1985) High efficiency gene transfer into mammalian cells. In DNA cloning. a practical approuch. ed. D. M. Clover, pp. 143~-190 (IRL Press Oxford). He X.. Treaty M. N.. Simmons D. M., lngraham H. A., Swmsno L. S. and Rosenfeld M. G. (1989) Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340, 35-42. Hill C. S., Wynne J. and Treisman R. (1994) Serum-regulated transcription by serum response factor (SRF): a novel role for the DNA binding domain. EMBO J. 13, 5421 5432. Lakin N. D.. Morris P. J.. Theil T., Sato T. N.. Moroy T.. Wilson M. C. and Latchman D. S. (1995) Regulation of neurite outgrowth and SNAP-25 gene ex-
factor
I199
pression by the Brn-3a transcription factor. .I Viol. Chem. 270, 15858-15X63. Lillycrop K. A., Budhram-Mahadeo V. S., Lakin N. D., Terrenghi G.. Wood J. N., Polak J. M. and Latchman D. S. (1992) A novel POU family transcription factor is closely related to Brn-3 but has a distinct expression pattern in neuronal cells. .vuc,/. A&Y Rcsv. 20, 5093 5096. Liu Y. Z.. Dawson S. J. and Latchman I>. S. i 14Q6) Alternative splicing of the Brn-3a and Brn-3b transcrlption factor mRNAs is regulated in neuronal cells ./. Mol. Neurosci. 7, 77.~85. McEvilly R. J.. Erkman L., Luo L.. Sawchenko P. E.. Ryan A. F. and Rosenfeld M. G. (1996) Requirement for Brn-3.0 in differentiation and survival of \cnsory and motor neurons. Nature 384, 574 577. Macpherson I. and Stoker M. (1962) Polyma tranxl’ornation of hamster cell clones- -an investigation 01‘ the genetic factors affecting cell competence. [‘irol~rr,’ 16, 147~151. Madden S. L.. Cook D. M. and Rauscher F. J. (1993) A structure-function analysis of transcriptional repression mediated by the WTI Wilms’ tumour suppressor protein. Onccqyw 8, 17 I3- 1720. Morgenstern J. P. and Land H. (1990) A series of mammalian expression vectors and Characterization 01’ their expression of a reporter gene in stably and tran\sently transfected cells. Ni,c/. Acids Rex. 18, 1068. Morris P. J.. Theil T., Ring C. J. A.. Lillycrop K. A,Miir
I500
Jo L. Begbie
and David
Wegner M.. Drolet D. W. and Rosenfeld M. G. (1993) POU-domain proteins: structure and function of developmental regulators. Curr. Opin. Cell Biol. 5, 488-498. Wood J. N., Bevan S. J., Coote P.. Darn P. M., Hogan P., Latchman D. S., Morrison C., Rougon G..
S. Latchman Theveniau M. and Wheatley S. C. (1990) Novel cell lines display the properties of nociceptive sensory neurons. Proc. Rovul Sot. London Serks B 241, 187 194.