(Received for publication, July 7, 1995; and in revised form, August 21, 1995)
From the
Using nuclear extracts prepared from rat liver it was demonstrated that binding of a transcription factor to site II of the D-site binding protein promoter could be induced by dephosphorylation of these extracts. Competition band shifts and supershift assays reveal this protein to be the general transcription factor Sp1. Phosphorylation of Sp1 appears to occur as a result of terminal differentiation of the liver. Proteins from both 1-day-old rat liver and adult liver undergoing regeneration have less of the phosphorylated form of Sp1 present with consequent increased DNA binding activity. Sp1 is similarly phosphorylated in brain, kidney, and spleen with phosphatase treatment of the extracts significantly increasing the level of DNA binding activity. Dephosphorylation of Sp1 results in a 10-fold increase in the affinity of Sp1 for its cognate site. Two-dimensional gel electrophoresis reveals that approximately 20% of the detectable protein appears to be in the phosphorylated form in adult liver extracts. Another protein with similar characteristics also appears to be present in the liver. Decreasing Sp1 DNA binding activity by phosphorylation may be an important mechanism for regulating gene expression, and possibly bringing about growth arrest during terminal differentiation.
The regulation of terminal differentiation is a complex process
involving both the induction of specific genes and growth arrest of the
differentiating cells. There are several model systems where the
expression of transcription factors involved in the induction of
differentiation are regulated by factors directly linked to the control
of the cell cycle(1, 2) . During myogenesis
interaction between Myo-DI, and the retinoblastoma protein appears to
be critical for bringing about, and maintaining, the terminally
differentiated state (3, 4, 5) . The
plasticity of differentiation in the liver, characterized by its
ability to undergo compensatory regenerative growth(6) ,
provides an in vivo model for the linkage of growth and
differentiation. Several liver enriched transcription factors including
CAAT/enhancer-binding protein, liver activator protein, and DBP ()are down-regulated in response to the induction of growth
and the loss of differentiation which occurs during liver
regeneration(7, 8) . Recently, the regulation of the
DBP gene, which encodes a member of the proline- and acidic rich domain
subfamily (9) of basic/leucine zipper proteins, has pointed to
another link between the cell cycle and differentiation. Factors
binding to the retinoblastoma control element (RCE) (10) were
found to be induced upon terminal differentiation and to bind to
several sites (site I and III) within the DBP proximal
promoter(11) . In contrast, the binding of proteins to site II
of the DBP promoter was found to be altered during differentiation.
Site II binds at least two proteins, one of which does not change
during differentiation. During the growth phase of regeneration the
presence of a slowly migrating complex was noted which is not present
in extracts from the normal liver. This pattern was paralleled by the
presence of a similar, slowly migrating complex in extracts from the
newborn liver. The reduction in DNA binding activity to site II in the
adult extracts appears to involve the well studied transcription factor
Sp1 and has suggested that Sp1 DNA binding activity is modified in
response to differentiation.
Sp1 is a member of the
C-H
zinc finger family (12) and was
one of the first transcription factors to be cloned(13) . It is
involved in the regulation of a wide variety of different genes,
including the early promoter of SV40(14) , genes involved in
proliferative response(15, 16, 17) ,
extracelluar matrix protein genes(18, 19) ,
housekeeping genes(20, 21, 22) , and a number
of growth factor genes (17, 23) . Sp1 has also been
implicated in the function of the RCE (24, 25) and
cotransfection of a retinoblastoma gene expression vector is able to
modulate the transactivation of responsive genes by Sp1. These findings
have linked Sp1 function to that of the retinoblastoma protein,
suggesting that Sp1 may be involved in modulating transcription in
response to the growth state of the cell.
Two different types of post-translational modification of Sp1 are known to occur. Sp1 contains a number of O-linked N-acetylglucosamine residues clustered in the amino-terminal half of the protein(26) . Glycosylation does not appear to affect the ability of this factor to bind DNA but the addition of soluble wheat germ agglutinin (WGA) does interfere with the transactivation of Sp1 responsive genes. Sp1 is also known to be phosphorylated by a DNA-dependent protein kinase which requires the presence of DNA ends for activity (27) and involves a large protein complex which includes the Ku antigen(28) . The phosphorylation of Sp1 by DNA-dependent protein kinase does not appear to alter the DNA binding activity of this protein.
Correlation of the changes in occupation of the DBP site II with the differentiation state of the liver and the observation that this site can act as a weak transcriptional activator led us to examine the molecular basis of this phenomenon. In this paper we have been able to demonstrate that Sp1 binds to site II of the DBP proximal promoter. Changes in protein-DNA complexes present on this site during differentiation led to the discovery that Sp1 DNA binding activity is modulated by phosphorylation. In the normal liver Sp1 is phosphorylated resulting in a decrease in its affinity for DNA. In de-differentiated tissues, such as regenerating or newborn rat liver, Sp1 is less phosphorylated and exhibits increased DNA binding activity. An additional factor, possibly Sp3, which also binds to an Sp1 recognition site but which is not recognized by an Sp1 antibody is also regulated in a similar manner. These results suggest that phosphorylation of Sp1, as well as closely related proteins, may be an important regulatory mechanism for controlling growth and differentiation in vivo.
Figure 1: Dephosphorylation of nuclear extracts induces DNA binding activity for site II and the RCE. Band shift assays were carried out with liver nuclear extracts, extracts treated with alkaline phosphatase, or extracts with phosphatase and various phosphatase inhibitors, as indicated. Oligonucleotides from DBP sites I-IV, the RCE, or the rat albumin C site which binds NF-Y, were used as probes. DNA complexed with proteins are indicated (BOUND) as is the position of the free DNA (FREE).
Figure 2: An Sp1 oligonucleotide competes for proteins binding to site II and the RCE and this activity is selected by WGA. A, band shift assays were carried out with the indicated probes and either normal or dephosphorylated nuclear extracts. Increasing amounts of unlabeled Sp1 oligonucleotide were added to the reactions as indicated. B, rat liver nuclear proteins were selected using WGA columns (Normal/WGA), followed by dephosphorylation (Dep/WGA), or proteins were first dephosphorylated and then selected with WGA (Dep/WGA). These proteins were used to carry out band shift assays with the Sp1 or site II probes as indicated.
Significantly less complex was formed between the dephosphorylated factor and the RCE in comparison to site II ( Fig. 1and Fig. 2A) suggesting that these sites might have different affinities for this factor. In order to compare their relative affinities, a band shift assay was carried out using dephosphorylated nuclear extracts selected on a WGA column with site II as the probe. Unlabeled DBP sites II and III, the RCE, and the Sp1 consensus site were then used as competitors for this activity. This revealed that site II and the consensus Sp1 site had comparable affinities with a maximum reduction in binding to 1 and 16%, respectively (Fig. 3A). The RCE was a very poor competitor in this assay with a maximal reduction in binding to only 75% of the starting value, only slightly better than the reduction to 91% with the unrelated site III. A similar experiment was carried out using purified Sp1 produced from a vaccinia virus expression vector, demonstrating that site II is a higher affinity binding site for Sp1 than the consensus Sp1 site (Fig. 3B). The RCE is a less efficient competitor than site II, with a maximal reduction in binding to 44%, however, it is a better competitor for recombinant Sp1 compared to the dephosphorylated WGA selected material suggesting that the WGA material may actually contain two different factors, one of which does not bind well to the RCE.
Figure 3: Sp1 binds to site II and weakly to the RCE. A, competition for WGA selected proteins binding to site II. Increasing amounts of the indicated unlabeled oligonucleotides were added to a band shift reaction containing WGA selected nuclear proteins and the site II probe. B, increasing amounts of the indicated unlabeled oligonucleotides were added to a band shift reaction containing recombinant Sp1 and the site II probe.
Figure 4: An Sp1 antibody does not recognize all of the site II or Sp1 site binding activity. Band shift assays with normal (Normal) or dephosphorylated (DEPHOSPH) nuclear extracts as well as recombinant Sp1 (rSp1) and the indicated probes were incubated with different amounts of an antibody to Sp1. The supershifted complex is indicated by an arrow (SS).
Figure 5: Sp1 phosphorylation is dependent on the differentiation state of the liver. Band shift assays were carried out with nuclear extracts from adult (NORMAL), regenerating (REGEN), or newborn (1 DAY) rat livers. The probe was the rat albumin C site (C) which recognizes NF-Y, or the Sp1 site (all others). Antibody to Sp1 was added in some reactions (Ab) and in others the extracts had first been dephosphorylated by the addition of alkaline phosphatase (DeP). The supershifted complex is indicated SUPERSHIFT.
Figure 6: Characterization of Sp1 phosphorylation by two-dimensional electrophoresis. Samples of normal adult rat liver nuclear extracts (RNLE), extracts from 1-day-old animals (1 DAY) and recombinant protein (rSp1) and the dephosphorylated forms of each (deP) were separated by isoelectric focusing in the first dimension and by SDS-polyacrylamide gel electrophoresis in the second. Proteins were then transferred to nitrocellulose filters and Sp1 protein detected using the Sp1 antibody and chemiluminescence.
Figure 7:
Phosphorylation associated with
differentiation decreases the affinity of Sp1 for its site. The K of Sp1 for its site in normal adult
liver (RNLE), regenerating liver (REGN), or
dephosphorylated adult liver (RNLE deP) extracts was measured
using band shift assays with increasing amounts of DNA to determine
saturation binding. Bound and free DNA were quantitated and bound DNA verses probe concentration for each condition is shown. The
derived K
and B
values, as well as the correlation coefficients, are indicated in
the table.
Figure 8: Sp1 is phosphorylated and its DNA binding activity is down-regulated in a variety of tissues. Nuclear extracts from the indicated tissues were used in band shift assays with the Sp1 probe either untreated(-) or after dephosphorylation (+).
Proteins binding to site II of the DBP promoter are subject
to differential regulation during terminal differentiation. In the
normal liver a ubiquitous factor binds to site II, the binding activity
of which does not appear to be altered during differentiation.
Competition experiments with various oligonucleotides indicate that
this factor is present in approximately equal quantities in the normal
or regenerating liver (results not shown). In the de-differentiated
liver Sp1 DNA binding activity is induced and binds to site II with an
affinity comparable to a consensus Sp1 site and significantly higher
than to the RCE. This is despite site II having more mismatches to the
derived Sp1 consensus sequence than either the Sp1 site or the RCE.
Additional experiments have indicated that Sp1 and the ubiquitous
factor have overlapping but distinct recognition sites and that binding
of these proteins is mutually exclusive. ()Site II in normal
liver nuclear extracts acts as a weak activator(11) .
De-differentiation results in the down-regulation of DBP transcription
apparently through changes in occupation of the proximal promoter
binding sites. This would suggest that Sp1 acts as a negative regulator
of site II activity. The binding of Sp1 to site II may result in the
displacement of the other factor and down-regulation of the
transcriptional activity of this site. Similar interactions are seen in
the human
-globin gene (33) where Sp1 competes with a
factor called stage selector protein for occupation of several sites.
The preferential binding of Sp1 is correlated with the inactivation of
this promoter.
There appear to be at least two proteins or Sp1 isoforms present in normal nuclear extracts which can be dephosphorylated and which bind to Sp1 sites. The supershift experiments failed to shift a significant portion of the activated proteins present. The Sp1 antibody used was a polyclonal antiserum directed against a peptide corresponding to amino acids 520 to 538 of human Sp1(14) . Because it recognizes a restricted epitope it is possible that post-translational modification of Sp1 blocks the ability of this antibody to recognize some of the Sp1 present. Alternatively, two new genes related to Sp1 have been isolated, one of which also recognizes Sp1 sites(32) . These genes, Sp2 and Sp3, have a low level of identity with the region of Sp1 used to produce the antibody (11/19 and 12/19 residues, respectively) and are likely not recognized in the supershift assay. In addition, the competition experiments with recombinant Sp1 and WGA selected proteins demonstrated that this other factor does not bind well to the RCE in comparison to Sp1. This further suggests that the binding activity observed corresponds to a distinct protein. Thus, another factor, which preliminary experiments suggest may be Sp3, is present in the liver and is phosphorylated upon terminal differentiation and while it recognizes Sp1 sites it appears to have some differences in its sequence specific recognition. Sp3 has recently been identified as a negative regulator of transcription (34) so that competition and differential regulation of Sp1 and Sp3 may be important for control of gene expression.
The phosphorylation of Sp1 during differentiation and the consequent decrease in its DNA binding activity suggests that this process is a significant regulatory mechanism during development. Sp1 has previously been shown to be phosphorylated by a DNA-dependent protein kinase(27, 28) , however, this modification does not effect the binding of Sp1 to a high affinity site. Differences in the DNA binding activity of Sp1 as assayed using Southwestern blot analysis have been previously reported(35) . Differences in DNA binding activity of a protein which has undergone denaturation and renaturation must be attributable to covalent modification rather than association of an inhibitory protein. Recently inhibitory proteins interacting with the amino-terminal part of Sp1 have been characterized(36, 37) . It is possible that the phosphorylation of Sp1 is linked to the binding of an inhibitory factor such as Sp1-I. The observation that significant amounts of non-phosphorylated Sp1 which does not appear to bind to DNA is present in the adult liver suggests that an additional level of control is also present.
The wide variety of genes which are regulated by Sp1 make
the significance of its differentiation-induced phosphorylation and
reduction in affinity particularly important. Many of the genes
regulated by Sp1 are required for cell growth and as such the
down-regulation of Sp1 binding activity upon terminal differentiation
would reduce the expression of these genes and possibly help to bring
about growth arrest. We have been able to demonstrate that the
serine/threonine kinase, casein kinase II, is capable of
phosphorylating the DNA binding domain of Sp1 and that this
phosphorylation results in a reduction in Sp1 DNA binding activity. ()It is possible that this kinase is responsible for the
observed phosphorylation of Sp1 during development. Casein kinase II
has been predicted to play a central role in the regulation of cellular
processes(38, 39) . Its ability to down-regulate the
activity of the AP-1 complex by phosphorylation of c-Jun, and the
increase of c-Jun phosphorylation in quiescent cells (40) suggests that this kinase is critical for modulating cell
growth. The concomitant need for growth arrest and differentiation
suggests that increased phosphorylation of both c-Jun and Sp1 by casein
kinase II is a critical step in bringing about terminal
differentiation. The demonstration that a transgenic mouse carrying an
extra copy of the casein kinase II
subunit (which appears to
disrupt casein kinase II function in some way) can produce a block to
differentiation resulting in a form of lymphoma (41) gives
further evidence that casein kinase II-mediated phosphorylation may
play an important role in bringing about growth arrest and
differentiation.
The retinoblastoma gene is known to be regulated by
Sp1 (42) and its expression is down-regulated during
differentiation(43) . However, other Sp1 regulated genes carry
out housekeeping functions and as such are required to be active in
terminally differentiated tissues. The general regulation in Sp1 DNA
binding activity would seem to preclude the expression of all Sp1
dependent genes in terminally differentiated tissues. Other
transcription factors are induced upon terminal differentiation and
could functionally compensate for the loss of Sp1 activity.
Alternatively, promoters which remain active after terminal
differentiation may have higher affinity binding sites for Sp1. This
might be an intrinsic property of the site, as appears to be the case
for the DBP site II, or protein-protein interactions could stabilize
Sp1 binding. Given that the change in K of Sp1 is
approximately 10-fold upon phosphorylation, significant binding to
promoters with high affinity sites would still be possible.
Phosphorylation of c-Myb by casein kinase II has been shown to affect
its binding to weak sites to a much greater degree than to strong sites (44) . This could provide for differential regulation of genes
where promoters with low, mid, and high affinity Sp1 sites are all
active in replicating, un-differentiated cells. Differentiation would
then result in the selective deactivation of promoters with lower
affinity sites which, if these genes were required for growth would
also bring about growth arrest. The high affinity promoters would
remain active under these circumstances and continue to produce
necessary proteins. This differentiation-induced phosphorylation of Sp1
does not appear to be unique to the liver as Sp1 activity can be
induced by phosphatase treatment in all adult tissues examined. In
addition, there does appear to be a correlation between the proportion
of actively replicating cells in a given tissue and the amount of Sp1
activity present. These observations suggest that alterations in the
phosphorylation state of Sp1 may be part of a ubiquitous mechanism
linking the regulation of growth and differentiation.