Download Inhibition of Pax 5 activity by expression of its DNA binding domain

BiochemicalSociety Transactions (1995) 23 325s
Inhibition of Pax 5 activity by expression of its DNA
binding domain.
G. Z. HASTINGS and B. ADAMS, .
Dept. Biochemistry, University of Sussex, BNI 9QG.
The B-cell specific transcription factor pax 5 encodes the Bcell specific activator protein (BSAP) and is expressed at all
stages of B-cell development except in terminally
differentiated plasma cells (I). Pax 5 has also been shown to
regulate the expression of B cell specific antigen CD19 (2).
This suggests that the expression of BSAP plays a role in Bcell commitment and development.
When a reporter plasmid containing Pax5 binding sites 5' of
a reporter gene is co-transfected with a plasmid expressing
Pax5, the reporter gene is not expressed. This suggests that
while Pax5 is necessary for transactivation of a target gene,
it is not sufficient (ie. a second B-cell specific factor must be
required). This conclusion is supported by the observation
that Pax5 is expressed in some non B-cell tissues
(developing CNS and the adult testis), while the B-cell
specific target gene. CD19, is not ( I ) . As the Pax5 gene
product is able to bind its target sequence in vitro, then the
seccond protein is likely to be involved not in binding DNA
but in transactivation by the DNA bound protein. Such a
factor would be expected to act in a similar way to the SAPI protein of the serum response Factor (3)
If this is true then a protein having the DNA binding
activity of Pax5, fused to the transactivation domain of a
ubiquitously active transactivator should activate
transactivation of the reporter gene. Such a construct is
described below and has been shown to transactivate a
reporter construct. I t is also expected that a protein
consisting of only the DNA binding domain, and no
transactivation sequences, will inhibit the transactivation of
the above protein. This has also been shown to be occur.
Reporter constructs contain the cDNA sequence of
chloramphenicol acetyl transferase (CAT). Located
upstream of the coding sequence a number of Pax 5 binding
sites are part of an artificial promoter. The two constructs
pBS4 and pBS2 contain 4 and 2 Pax 5 binding sites
respectively. pBSMN contains one mutant (non-functional)
binding site followed by a functional site and pBSNM
contains a functional Pax 5 binding site followed by a
mutant site. Three Pax 5 expression vectors were used.
phBSAP.VP16 encodes a fusion protein, in which the Pax 5
DNA binding domain is linked to the VP16 transactivation
domain (4). phBSAPls encodes the native Pax 5 gene
product. pCI encodes a truncated Pax 5 gene product
having only the DNA binding activity.
1 00
80
3
5L
60
8
40
E 2 ug inhib
10 ug inhib
b?
I
20 ug inhib
20
0
PCI
phBSAP
Fig. 2. Inhibition ofphBSAP.VP16 by the native Pux 5
gene product, and the isolated DNA binding domuin. 5ug
of reporter construct pBS2 und 2pg ofphBSAP.VP16 were
co-transfected with increasing amounts of either pCI or
phBSAPls. CAT expression w m determined us described
for Fig. I .
is obtained if these constructs are co-transfected with
phBSAPl s. This confirms the previous observation that the
Pax 5 gene product is not sufficient to activate target genes.
Co-transfection of the reporter constructs with
phBSAP.VP16 results in the expression of CAT activity
(Fig. I). The level of expression is dependant both on the
number of Pax 5 binding sites and the amount of the
expression vector used. The replacement of the Pax 5 gene
product's C-terminal domain with the VPI 6 transactivation
domain has resulted in a transcription factor which is
independent of a B-cell background. This is consistent with
it no longer requiring a specific co-protein. When both
phBSAP.VP16 and pC1 are co-transfected with a reporter
construct, the level of CAT expression is reduced (Fig 2).
As more pCI is included in the tranfection, the level of CAT
expression is competed out. An identical result is obtained
if PhBSAPIs is used instead of pCI. This suggests that the
Pax 5 DNA binding domain is acting as a competitive
in hi bi tor.
The data described above are consistent with the model that
although the Pax 5 gene product is essential for the
activation of its target gene, it is not sufficient. That
transactivation of a target gene occurs when the Pax 5 gene
products C-terminal domain is replaced with an alternative
transactivation domain, shows that the inability of Pax 5 to
function as a cell-type independent transcription factor is
due to a B-cell specific co-protein required to interact with
the C-terminal transactivation domain.
Both the isolated Pax 5 gene product's DNA binding
domain and the native protein competitively inhibit
transactivation bv ~ h B S A P . v p l 6 . The onlv reasonable
binding to these target sequences.
7t,
.
We thank Dr. Kevin Lee for providing the Pax 5 reporter
constructs. Experiments described in this report were
supported by funds from the Leukaemia Research Fund.
pBSMN
pBSNM
pBS2
pBS4
F i g . 1. Activution of Pux 5 reporter constructs by
phBSAP. VP16. Reporter pkusmids were transfected into
HeLu cells. either with or without phBSAP.VP16, b y
electroporation. After 24 his. cell extracts were mude.
CAT uctivity determined und the % conversion culculated
us described (5).
I . Adams,B., Dorfler,P., Aguzzi,A., Kozmic,Z., and
Busslinger,M. 1992, Genes and Dev. 6, 1589-1607.
2. Kozmic,Z., WongS., Dorfler,P., Adams,B., and
Busslinger,M. 1992, Mol. Cell. Biol. 12, 2662-2672.
3. Dalton,S. and Treisman,R. 1992, Cell 68,5!37-612
4. SadowskiJ., BelLB., Br0ad.P. and Hol1is.M. 1992, Gene
116,137-141.
5. Buschle,M., Brenner,M., Chen,I., Drex1er.H.. Gignac.S. and
Rooney,C. 1990. J. Immunol. Meth. 133,77-85.