buttonhead
D-Sp1, whose DNA binding domain is more closely related to Sp1 than to Buttonhead, maps to the same cytogenetic location as btd, band 9A of the X-chromosome. Thus, btd and D-Sp1 may represent a pair of genes with partially overlapping functions. Drosophila Sp1 is expressed in btd-like patterns during post-blastodermal stages of embryogenesis. However the early expression patterns of the two genes are different. Low levels of D-Sp1 transcripts are found evenly distributed throughout the preblastoderm embryo, suggesting that D-Sp1 is expressed maternally. At blastoderm, when btd is expressed in the head stripe domain and the dorsal spot, no corresponding D-Sp1 transcripts are observed. First zygotic expression of D-Sp1 occurs in the proneural region of the head during early gastruation and expression continues in the corresponding domain until the germ band is fully extended. From then on, the expression patterns of the two genes are similar, except that the metameric pattern of D-Sp1 expression is much weaker than observed with btd (Wimmer, 1996).
Buttonhead encodes a
zinc-finger-type transcription factor with sequence and functional similarity to mammalian transcription factor Sp1. The transcriptional activation domain of Sp1 has two serine/threonine-rich domains, alternating with two glutamine-rich domains. The Btd box is found in five out of six members of the vertebrate Sp1-related family. When expressed in the spatial pattern of BTD, a
transgene providing Sp1 activity can support development of the mandibular segment in the head of
btd mutant embryos (Wimmer, 1993).
Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. The ortholog of the Drosophila Sp-1 gene has been identified in the red flour beetle Tribolium castaneum and has been termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8RNAi legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8RNAi leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, it is hypothesized that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth (Beermann, 2004).
The T-Sp8 protein is considerably shorter than the Drosophila ortholog (691 amino acids) because of the lack of the long glycine and serine repeats in the N-terminal and the glutamine-rich regions in the C-terminal part of the protein. An alignment
of the amino acid sequences from these two species reveals high conservation not only of the zinc-finger region but also of two other protein motifs in the Sp genes, the Sp-box and the Buttonhead (Btd)-box. The Sp-box is a 13 amino acid motif located at the N terminus of the protein, whereas the Btd-box has been described as a 10 amino acid box situated immediately N terminal to the zinc-finger domain. At the moment, the roles of these motifs for the function of the Sp protein remain unknown. They are suggested to be involved in transactivation activity (Btd-box) or as an interaction-site with a repressor (Sp-box) (Bouwman,
2002). The comparison to the D-Sp1 sequence reveals a
conserved stretch of 43 amino acids in the Sp-box region of both insect
species that includes the described Sp-box core. The alignment of the
Sp-box to the vertebrate orthologs shows that T-Sp8 is slightly more similar to the as yet functionally uncharacterized human SP8 gene than to the osterix/Sp7 gene that
has been shown to be required for bone development in the mouse. The Btd-box of Tribolium-Sp8 , Drosophila Sp1 and human SP8 is
identical and 2/10 amino acids are different in the Btd-box of human
SP7 (Beermann, 2004 and references therein).
Little is known about the factors that control the specification of the mid-hindbrain domain (MHD) within the vertebrate embryonic neural plate. Because the head-trunk junction of the Drosophila embryo and the MHD have patterning similarities, vertebrate genes have been sought related to the Drosophila head gap gene buttonhead (btd), which in the fly specifies the head-trunk junction. The identification of a zebrafish gene which, like btd, encodes a zinc-finger transcriptional activator of the Sp-1 family (hence its name, bts1 for btd/Sp-related-1) is reported; bts1 shows a restricted expression in the head. During zebrafish gastrulation, bts1 is transcribed in the posterior epiblast, including the presumptive MHD, and precedes in this area the expression of other MHD markers such as her5, pax2.1 and wnt1. Ectopic expression of bts1 combined to knock-down experiments demonstrates that Bts1 is both necessary and sufficient for the induction of pax2.1 within the anterior neural plate, but is not involved in regulating her5, wnt1 or fgf8 expression. These results confirm that early MHD development involves several genetic cascades that independently lead to the induction of MHD markers, and identify Bts1 as a crucial upstream component of the pathway selectively leading to pax2.1 induction. In addition, they imply that flies and vertebrates, to control the development of a boundary embryonic region, have probably co-opted a similar strategy: the restriction to this territory of the expression of a Btd/Sp-like factor (Tallafuss, 2001).
Tail bud stage wild-type zebrafish cDNA was PCR-amplified using degenerate oligonucleotides directed against the zinc-finger
domains of Btd and Sp factors. Eleven partial cDNAs encoding zinc finger domains were obtained, each of them from
several distinct PCR reactions, suggesting that they correspond to different genes and not to variations due to Taq polymerase
errors. All code for triple zinc fingers, 55-85% similar to each other and with the structure Cys2-His2 characteristic of Btd and Sp
factors. They were named bts genes (for btd/Sp-related). Except in two cases (g5.6 and g5), they are more closely related to the zinc-finger domain of Sp factors
(70%-94% identity) than to that of Btd (64%-80% identity). g5.6 is equally related to Sp and Btd (75% identity), and g5 is more closely related to Btd than to Sp (69% versus 56% identity) (Tallafuss, 2001).
To determine whether one of these factors could be a functional equivalent of Btd at the Drosophila head-trunk junction, their expression profiles were examined at the tail bud stage using high-stringency whole-mount in situ hybridization conditions. With the exception of g5.6 and G1, which proved ubiquitously expressed, all other genes tested showed spatially restricted and distinct expression patterns, further confirming that they corresponded to different factors. One of them, bts1, appears to be selectively expressed in the MHD, and was therefore selected for further studies. g5, the most related in sequence to btd, was not expressed in the mid-hindbrain and thus appeared unlikely to be a functional homolog of btd in this domain (Tallafuss, 2001).
These experiments have allowed a test of the hypothesis that factors expressed at the Drosophila head-trunk and vertebrate mid-hindbrain junctions would be conserved during evolution. This hypothesis was based on previous reports that documented the expression of homologous genes of the otd/Otx, engrailed/En and pax2/5/8 families at equivalent AP levels in urochordate, vertebrate and insect embryos. Bts1 and Btd do share some functional characteristics, since Bts1 could rescue the expression of col in a correct spatiotemporal manner in btd mutants. Bts1 is neither capable of rescuing the expression of eve and en nor the formation of posterior head structures in btd mutants. Under similar conditions, Sp1 can partially restore en expression and mandibular derivatives. Since a chimeric protein composed only of the SP1 zinc finger fused to the activation domain of VP16 also rescues en expression, and given the conservation of Bts1 and Sp1 zinc fingers, Bts1 might simply not have sufficient activity to transactivate the en promoter. A similar hypothesis might hold true for the failure of both Bts1 and Sp1 to sustain the development of intercalary and antennal segments. Alternatively, in these processes, Btd might need to interact with cofactors incapable of recognizing the divergent non DNA-binding modules of Bts1 and Sp1 (Tallafuss, 2001).
Taken together, these results indicate that Btd and Bts1 share expression and function characteristics in their control of the development of a comparable boundary region of the embryo. btd and bts1 might have diverged from a common ancestor involved in the development of posterior head territories, or might have been co-opted during evolution in the fly and in vertebrates. The second hypothesis is favored, since Bts1 is more related in sequence to the extant subfamily of Sp factors, including Drosophila Sp1, than to the Btd subfamily (which comprises zebrafish members such as the clone g5). These results therefore have interesting evolutionary implications because they strongly suggest that flies and vertebrates, by restricting to the head-trunk or mid-hindbrain junction the expression and functional domain of a Btd/Sp-family member, have independently developed a similar strategy to pattern comparable territories. Whether Bts1 and Btd are part of a conserved molecular cascade awaits further analysis; it is noted, for example, that col has no vertebrate homolog expressed at the mid-hindbrain junction (Tallafuss, 2001).
Mouse sp4, expressed in the developing brain, is the closest identified vertebrate homolog of Buttonhead. Earliest expression at embryonic day 9 is highest in the region surrounding the posterior neuropore. In the day 10 embryos, the strongest staining is observed in the developing brain and neural tube. By day 13, expression is detected in other neural structures, such as the retina, trigeminal ganglia, spinal ganglia, and developing cranial nerves. In addition to the nervous system, SP4 is found in the nasal mucosa and the vomeronasal organ, Rathke's pouch, the hypothalamic region, hepatocytes in the liver, mucosal epithelium of the intestinal tract, bronchial epithelium of the lung and the tubules of the metanephric kidney. Knockouts are born with no apparent limb, nervous system, or craniofacial malformations, but never nurse and die within 24 hours after birth (Supp, 1996).
Sp1 is a sequence-specific DNA binding protein that activates RNA polymerase II transcription from promoters that contain properly positioned GC boxes. A series of deletion mutants of Sp1 were expressed in Escherichia coli and used to identify separate regions of the protein that are important for three different biochemical activities. The sequence-specificity of DNA binding is conferred by Zn(II) fingers, whereas a different region of Sp1 appears to regulate the affinity of DNA binding. E. coli-synthesized Sp1 is able to stimulate initiation of RNA synthesis in vitro, and at least two distinct segments of the protein contribute to its transcriptional activity (Kadonaga, 1988).
Initiation and maintenance of signaling centers is a key issue during
embryonic development. The apical ectodermal ridge, a specialized epithelial
structure and source of Fgf8, is a pivotal signaling center for limb
outgrowth. Two closely related buttonhead-like
zinc-finger transcription factors, Sp8 and Sp9, are
expressed in the AER, and regulate Fgf8 expression and limb
outgrowth. Embryological and genetic analyses have revealed that Sp8
and Sp9 are ectodermal targets of Fgf10 signaling from the mesenchyme. Wnt/ß-catenin signaling positively regulates Sp8, but not Sp9. Overexpression functional analyses in chick unveiled Sp8
and Sp9 role as positive regulators of Fgf8
expression. Moreover, a dominant-negative approach in chick and knockdown
analysis with morpholinos in zebrafish revealed Sp8
and Sp9 requirement for
Fgf8 expression and limb outgrowth, and further indicate that Sp8
and Sp9 have a coordinated action on Fgf8 expression. This study demonstrates that Sp8 and Sp9, via Fgf8, are involved in
mediating the actions of Fgf10 and Wnt/ß-catenin signaling during
vertebrate limb outgrowth (Kawakami, 2004).
Murine Sp5 encodes a
protein having a C-terminal C2H2 zinc finger domain closely related to that of the transcription factor Sp1. In vitro, DNA
binding studies show that Sp5 binds to the GC box, a DNA motif present in the promoter of a very large number of genes,
including Brachyury, and recognized by members of the Sp1 family. However, outside of its DNA binding domain, Sp5 has
little homology with any other member of the Sp1 family. In contrast to the ubiquitous expression of Sp1, Sp5 exhibits a
remarkably dynamic pattern of expression throughout early development. This is suggestive of a role in numerous tissue
patterning events, including gastrulation and axial elongation; differentiation and patterning of the neural tube, pharyngeal
region, and somites; and formation of skeletal muscle in the body and limbs. Mice homozygous for a targeted mutation in
Sp5 show no overt phenotype. However, the enhancement of the T/1 phenotype in compound mutant mice (Sp5lacZ/Sp5lacZ,T/1) indicates a genetic interaction between Sp5 and Brachyury. These observations are consistent with a role for Sp5 in
the coordination of changes in transcription required to generate pattern in the developing embryo (Harrison, 2000).
buttonhead (btd) encodes an SP1-like transcription factor required for the generation and specification of Drosophila head segments. A murine btd homolog, termed mouse Btd (mBtd), has been identified, which can support btd-dependent head development in transgenic fly embryos. Functional studies show that mBtd-deficient mice develop to term and die at birth. They exhibit brain malformations, posterior axial skeleton truncations, and shortened limbs. Evidence is presented that mBtd is required during early limb development to maintain, but not to initiate Wnt/ß-catenin-dependent FGF, Shh, and BMP-mediated signaling. The data indicate that mBtd represents a novel key player mediating proximodistal outgrowth of the limb (Treichel, 2003).
The putative protein sequence of the btd-like gene reveals an SP1-like zinc finger and buttonhead domain. Thus, btd-like gene is a member of the SP1 family of transcription factors, such as SP1 and SP4 in mouse and human, D-SP1 and BTD in Drosophila, and BTS-1 in zebra fish. The highly conserved zinc finger domain (>90% sequence identity) is insufficient to reconstruct the evolutionary relationship of the btd-like gene to other members of this gene family. Therefore, a comparison of the entire proteins was undertaken. The putative protein contains a serine/threonine domain followed by an alanine-rich sequence similar to D-SP1 and BTD. It also contains a glycine-rich domain, which is found in D-SP1, the paralog of BTD. In contrast, the glutamine-rich sequence found in BTD, D-SP1, SP1, SP4, and BTS1 is absent in the putative protein. This domain in BTD and D-SP1 cannot account for an evolutionary link to BTS1, since this sequence rather represents a typical opa-element, which is found in nonhomologous genes in Drosophila. Accordingly, it is proposed that Bts1 is an ortholog of Sp1 and Sp4, whereas the newly identified gene represents an ortholog of D-Sp1 and btd (Treichel, 2003).
To test in vivo for evolutionary conserved functions of btd homologs, different genes were expressed under the control of the regulatory region of the btd gene in btd mutant Drosophila embryos. In these rescue experiments, the two previously identified btd-like genes Sp1 and Bts1 failed to provide any scorable rescuing activity anterior to the mandibular segment. The expression of one copy of the newly identified gene results in the development of btd-dependent mandibular segments (100%) and, in addition, intercalary segments. Therefore, the encoded protein contains functional features of BTD, which are absent from SP1 and BTS1. In analogy to the Drosophila gene btd, the gene has been termed mouse Btd (mBtd) (Treichel, 2003).
The spatiotemporal aspects of mBtd expression were analyzed by whole-mount in situ hybridization. Initial expression of mBtd is found between embryonic day 7.0 (E7.0) and E7.5 of gestation, with a strong hybridization signal during gastrulation in embryonic ectoderm and primitive streak. During secondary gastrulation, transcripts become restricted to the tail bud. During organogenesis, mBtd mRNA appears within the central nervous system in the telencephalon, midbrain-hindbrain boundary (MHB), spinal cord, otic vesicles, and nasal placodes (Treichel, 2003).
Outside of the CNS, mBtd activity is restricted to the limbs. It is initially observed in the entire ectoderm of the limb anlagen at E9.5, with a more abundant expression in the ventral part. The expression becomes progressively more prominent in the AER and is additionally detected as a clear but less prominent signal in the ventral ectoderm (Treichel, 2003).
These patterns suggest a possible role of mBtd during gastrulation and during CNS and limb development. To elucidate the role of mBtd during mouse embryogenesis, knockout experiments were performed using a homologous recombination approach in which the coding region of mBtd was replaced by a bacterial lacZ gene in embryonic stem cells. Heterozygous mBtd mutant individuals develop to term. About one-third of them exhibit a slight kink at the tip of the tail, suggesting that a reduction of the mBtd gene dose impairs posterior vertebra development at low but significant penetrance. Homozygous mBtd-deficient embryos also develop to term and die either during birth or immediately thereafter (Treichel, 2003).
Morphological and histological inspection of the homozygous mBtd mutant individuals revealed multiple and severe developmental defects in locations where the gene normally is expressed. Exencephaly at the level of the forebrain, an acerebellar hindbrain, spina bifida, and loss of nasal and palate structures were observed in these embryos. Most striking were truncations of the posterior axis and a reduction of both fore- and hind-limbs (Treichel, 2003).
Sp1, a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in
the whole animal; this results in decreased DNA binding activity. Casein kinase II (CKII) is able to phosphorylate the C
terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CKII may be responsible for the observed
regulation of Sp1. Mutation of a consensus CKII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of
this site and the CKII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CKII site at Thr-579 as
well as additional sites within the C terminus. No gross changes in CKII subunit levels were seen during de-differentiation associated
with liver regeneration. The serine/threonine phosphatase PP1 is identified as the endogenous liver nuclear protein able to
dephosphorylate Sp1 but again no gross changes in activity are observed in the regenerating liver. Okadaic acid treatment of K562
cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are
established by a balance between kinase and phosphatase activities (Armstrong, 1997).
The pituitary peptide hormone ACTH regulates
transcription of the cholesterol side chain cleavage cytochrome P450
(CYP11A) gene via cAMP and activation of
cAMP-dependent protein kinase. A G-rich sequence element
conferring cAMP-dependent regulation has been found to
reside within region -118 to -100 of the bovine CYP11A
promoter. This region has been shown to bind a protein
antigenically related to the transcription factor Sp1. The -118/-100 element binds both Sp1 and Sp3, members of the Sp
family of transcription factors.
Drosophila SL2 cells, which lack endogenous Sp factors, were used to
dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors
stimulate the activity of cotransfected reporter constructs in which
the promoter of the bovine CYP11A gene regulates luciferase
expression. Sp3 does not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of
the -118/-100 element of CYP11A abolishes Sp1-mediated
activation of a CYP11A reporter gene in SL2 cells as well
as cAMP responsiveness in human H295R cells. Furthermore,
cotransfection of SL2 cells with the catalytic subunit of
cAMP-dependent protein kinase, together with Sp1 and a
CYP11A reporter construct, enhances
Sp1-dependent activation of the reporter 4.2-fold,
demonstrating that Sp1 confers cAMP responsiveness in these cells.
Thus, introduction of Sp1 alone in an Sp-negative cell
such as SL2 is sufficient to achieve the cAMP-dependent
regulation observed using the -118/-100 element of CYP11A
in adrenocortical cells (Ahlgren, 1999).
The mechanism by which Sp1 confers cAMP responsiveness to the CYP11A promoter is not yet defined. Tissue-specific and/or hormonal regulation of Sp1
expression is a possible mechanism by which Sp1 activity could be modulated. However, the levels of Sp1 protein in adrenocortical cells or ovarian
granulosa cells are not altered by forskolin treatment as determined by immunoblot analysis. This suggests that other mechanisms, such as post-translational
modifications of Sp1 or regulated interactions of Sp1 with other factors, are more likely to be involved. Sp1 has been shown to be a phosphoprotein, but the effect
of phosphorylation on Sp1 activity is complex. Phosphorylation by a DNA-dependent kinase appears not to alter the activity, whereas casein kinase
II-mediated phosphorylation results in a decreased DNA binding activity. Of particular interest is the finding that PKA phosphorylation of Sp1 in vitro
increases its DNA binding activity. However, when the -118/-100 element was employed as a probe in EMSA, the same gel shifts were
obtained with nuclear extracts from untreated or forskolin-treated adrenocortical cells, indicating that forskolin does not affect the DNA binding activity of Sp1 in this
cell type for this particular site. Thus, although Sp1 can serve as a substrate for PKA phosphorylation in vitro, it has not yet been demonstrated
that this occurs in vivo in adrenocortical or other steroidogenic cells. It is recognized that transcription may be activated through the interaction of Sp1 with several other transcription factors, including CCAAT/enhancer-binding protein, p53, the chicken
ovalbumin upstream promoter transcription factor, and the estrogen receptor. Increasing evidence is emerging that Sp1
enhances cooperative interactions among multiple transcription factors to juxtapose the transcriptional regulatory domains of the proteins with the transcription
initiation complex. Whether similar cooperative interactions of Sp1 with other transcription factors underlie the cAMP responsiveness of CYP11A remains
to be determined (Ahlgren, 1999 and references).
Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. Cyclin A activity is regulated on
several levels and cyclin A elevation in a number of cancers suggests a role in tumorigenesis. In the present study, a modified DNA binding site selection and PCR amplification procedure was used to identify DNA binding proteins that are potential substrates of cyclin A-CDK. One of the sequences identified is the Sp1 transcription factor binding site. Co-immunoprecipitation experiments show that cyclin A and Sp1 can interact physically. In vitro and in vivo phosphorylation studies indicate that cyclin A-CDK complexes can phosphorylate Sp1. The phosphorylation site is located in the N-terminal region of the protein. Cells overexpressing cyclin A have elevated levels of Sp1 DNA binding activity, suggesting that cyclin A-CDK-mediated phosphorylation augments Sp1 DNA binding properties. In co-transfection studies, cyclin A expression stimulates transcription from an Sp1-regulated promoter. Mutation of the phosphorylation site abrogates cyclin A-CDK-dependent phosphorylation, augmentation of Sp1 transactivation function and DNA binding activity (Borja, 2001).
Members of the transforming growth factor-beta superfamily mediate a
broad range of biological activities by regulating the expression of target
genes. Smad proteins play a critical role in this process by binding directly to
the promoter elements and/or associating with other transcription factors.
TGF-beta1 up-regulates several genes transcriptionally through Sp1 binding
sites; however, the mechanism of TGF-beta induction of gene expression through
Sp1 sites is largely unknown. A novel 38-base pair TGF-beta-responsive element has been identified in the human plasminogen activator
inhibitor-1 (PAI-1) promoter, which contains two Sp1 binding sites, and is
required for TGF-beta-induced Smad-dependent transcriptional activation. Three
canonical Sp1 binding sites also support strong transcriptional activation by
TGF-beta and Smads from a minimal heterologous promoter. TGF-beta induction of
PAI-1 and p21 is blocked by the Sp1 inhibitor mithramycin, implicating Sp1 in
the in vivo regulation of these genes by TGF-beta. The association
between endogenous Sp1 and Smad3 is induced by TGF-beta in several cell lines;
however, Smad4 shows constitutive interaction with Sp1. These data provide novel
insights into the mechanism by which TGF-beta up-regulates the expression of several genes
by activating Sp1-dependent transcription through the induction of
Smad/Sp1 complex formation (Datta, 2000).
Sp1 activates the transcription of many cellular and viral genes with the GC-box in either the proximal promoter or the enhancer. Sp1 is composed of several functional domains, such as the inhibitory domain (ID), two serine/threonine-rich domains, two glutamine-rich domains, three C2H2-type zinc finger DNA binding domains (ZFDBD), and a C-terminal D domain. The ZDDBD is the most highly conserved domain among the Sp-family transcription factors and plays a critical role in GC-box recognition. In this study, the protein-protein interactions occurring at the Sp1ZFDBD, and the Sp1ID and the molecular mechanisms controlling these interactions, were examined. The results found that Sp1ZFDBD and Sp1ID repress transcription once they are targeted to the proximal promoter of the pGal4 UAS reporter fusion gene system, suggesting molecular interaction with the repressor molecules. Indeed, mammalian two-hybrid assays, GST fusion protein pull-down assays, and co-immunoprecipitation assays showed that Sp1ZFDBD and Sp1ID are able to interact with corepressor proteins such as SMRT, NcoR, and BCoR. The molecular interactions appear to be regulated by MAP kinase/Erk kinase kinase (MEK). The molecular interactions between Sp1ID and the corepressor might explain the role of Sp1 as a repressor under certain circumstances. The siRNA-induced degradation of the corepressors resulted in an up-regulation of Sp1-dependent transcription. The cellular context of the corepressors and the regulation of molecular interaction between corepressors and Sp1ZFDBD or Sp1ID might be important in controlling Sp1 activity (Lee, 2005).
Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, it has been demonstrated that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, can not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters (Smale, 1990).
The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription. The isolation and characterization of the first gene encoding a TAF protein is described. The deduced amino acid sequence of TAF110 reveals the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization. In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1. Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro. These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex (Hoey, 1993).
Activation of transcription by the promoter-specific factor Sp1 requires coactivators that are tightly associated with the TATA-box-binding protein (TBP) in the TFIID complex. Recent work has shown that the two glutamine-rich activation domains (A and B) of Sp1 can interact with at least one component of this complex, the TBP-associated factor dTAFII110. A region of Sp1 with alternating glutamine and hydrophobic residues, which is required for the interaction with dTAFII110 and is important for mediating transcriptional activation, has been mapped. Substitution of bulky hydrophobic residues within this region decreases both interaction with dTAFII110 and transcriptional activation in Drosophila cells. In contrast, mutation of glutamine residues in this region has no effect. Thus, the strength of the Sp1-TAF interaction correlates with the potency of Sp1 as a transcriptional activator, indicating that this activator-TAF interaction is an important part of the mechanism of transcriptional activation. Sequence comparison of three activation domains shown to bind dTAFII110 suggests that different activators that utilize dTAFII110 as a coactivator may share common sequence features that have been determined to be important for the Sp1-dTAFII110 interaction (Gill, 1994).
Transcriptional regulators can bind selected TAF subunits of the TFIID complex. However, the specificity and function of individual TAFs in mediating transcriptional activation remains unknown. The in vitro assembly and transcriptional properties of TBP-TAF complexes reconstituted from the nine recombinant subunits of Drosophila TFIID are reported. A minimal complex containing TBP and TAFII250 directs basal but not activator-responsive transcription. By contrast, reconstituted holo-TFIID supports activation by an assortment of activators. The activator NTF-1, which binds TAFII150, stimulates transcription with a complex containing only TBP, TAFII250, and TAFII150, whereas Sp1 binds and additionally requires TAFII110 for activation. Interestingly, TAFII150 enhances Sp1 activation even though this subunit does not bind directly to Sp1. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggest that TAFs perform multiple functions during activation (Chen, 1994).
Transcription factor Sp1, a zinc finger protein with three zinc finger C2H2 motifs, has been implicated in the expression of many genes. The protein is phosphorylated and highly glycosylated. The N-terminus contains glutamine- and serine/threonine-rich domains that are essential for transcriptional activity. The C-terminal domain of Sp1 is involved in synergistic activation and interaction with other transcription factors. Sp1 has been shown to interact directly with the TATA-box protein accessory factor TAFII110. When bound to distant sites in cis, it can interact with itself, thus looping out the intervening DNA. This suggests that Sp1 may establish interactions between promoters and distant regulatory elements in vivo through such a looping mechanism. It has been
suggested that Sp1 is linked to the maintenance of methylation-free CpG islands, the cell cycle, and the
formation of active chromatin structures. Sp1 knockout embryos
are retarded in development, show a broad range of abnormalities, and die around day 11 of gestation.
In Sp1-/- embryos, the expression of many putative target genes, including cell cycle-regulated genes, is
not affected; CpG islands remain methylation free, and active chromatin is formed at the globin loci.
However, the expression of the methyl-CpG-binding protein MeCP2 is greatly reduced in Sp1-/-
embryos. MeCP2 is thought to be required for the maintenance of differentiated cells. It is suggested that
Sp1 is an important regulator of this process (Marin 1997 and references).
Activation of gene transcription in metazoans is a multistep process that is triggered by factors that
recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct
transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II)
subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize
core promoter sequences necessary for transcription initiation. Transcriptional activation by
enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has
remained unknown. A new human factor, CRSP, is described that is required together with the
TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of
approximate relative molecular mass 700,000 [M(r) approximately 700K] that contains nine subunits
with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that
CRSP33 is a homolog of the yeast mediator subunit Med7, whereas CRSP150 contains a domain
conserved in yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear hormone-receptor
co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of
CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these
subunits have an essential cofactor function. The presence of common subunits in distinct cofactor
complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional
activation (Ryu, 1999).
Are transcription factors with diverse DNA binding domains able to exploit nucleosome disruption by SWI/SNF? To test this, an investigation was made into the possible mechanisms by which the SWI/SNF complex differentially regulates different genes. In addition to GAL4-VP16, the SWI/SNF complex
stimulates nucleosome binding by the Zn2+ fingers of Sp1, the basic helix-loop-helix domain of USF,
and the rel domain of NF-kappaB. In each case SWI/SNF action results in the formation of a stable
factor-nucleosome complex that persists after the detachment of SWI/SNF from the nucleosome. Thus,
stimulation of factor binding by SWI/SNF appears to be universal. The degree of SWI/SNF stimulation
of nucleosome binding by a factor appears to be inversely related to the extent that binding is inhibited
by the histone octamer. Cooperative binding of 5 GAL4-VP16 dimers to a 5-site nucleosome enhances
GAL4 binding relative to a single-site nucleosome, but this also reduces the degree of stimulation by
SWI/SNF. The SWI/SNF complex increases the affinity of 5 GAL4-VP16 dimers for nucleosomes
equal to that of DNA but no further. Similarly, multimerized NF-kappaB sites enhance nucleosome
binding by NF-kappaB and reduce the stimulatory effect of SWI/SNF. Thus, cooperative binding of
factors to nucleosomes is partially redundant with the function of the SWI/SNF complex (Utley, 1997).
Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. This study investigated the mechanism(s) through which cyclin D1 represses Sp1. The ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s), was examined. The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and, recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduces the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) is sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250 (Adnane, 1999).
Sequence-specific DNA-binding activators, key regulators of gene expression, stimulate transcription in part by targeting the core promoter recognition TFIID complex and aiding in its recruitment to promoter DNA. Although it has been established that activators can interact with multiple components of TFIID, it is unknown whether common or distinct surfaces within TFIID are targeted by activators and what changes if any in the structure of TFIID may occur upon binding activators. As a first step toward structurally dissecting activator/TFIID interactions, the three-dimensional structures of TFIID bound to three distinct activators (i.e., the tumor suppressor p53 protein, glutamine-rich Sp1 and the oncoprotein c-Jun) was determined and their structures were compared as determined by electron microscopy and single-particle reconstruction. By a combination of EM and biochemical mapping analysis, these results uncover distinct contact regions within TFIID bound by each activator. Unlike the coactivator CRSP/Mediator complex that undergoes drastic and global structural changes upon activator binding, instead, a rather confined set of local conserved structural changes were observed when each activator binds holo-TFIID. These results suggest that activator contact may induce unique structural features of TFIID, thus providing nanoscale information on activator-dependent TFIID assembly and transcription initiation (Liu, 2009).
Three D density difference maps generated from reconstructions of the three independent activator/TFIID assemblies (i.e., p53-IID, Sp1-IID, and c-Jun-IID) and free holo-TFIID have served as a method to map the most likely contact sites of these activators within the native TBP-TAF complex. Remarkably, each activator contacts TFIID via select TAF interfaces within TFIID. The unique and localized arrangements of these three activators contacting different surfaces of TFIID could be indicative of the wide diversity of potential activator contact points within TFIID that would be dependent on both the specificity of activation domains as well as core promoter DNA sequences appended to target gene promoters. It is also possible, however, that these distinct activator-TFIID contacts can form a common scaffold when TFIID binds to the core promoter DNA (Liu, 2009).
It is well established that activators including p53, Sp1, and c-Jun frequently work synergistically with each other or other activators to potentiate selective gene expression programs in response to a variety of stimuli in vivo. Therefore, combinatorial mechanisms of promoter activation might favor distinct nonoverlapping activator-binding sites within TFIID, which can be achieved by specific interactions between selective TAF subunits and activators. Indeed, it was established that TAF1 and TAF4 serve as coactivators for Sp1, while TAF1, TAF6, and TAF 9 mediate p53-dependent transactivation and TAF1 and TAF7 subunits are thought to be coactivators for c-Jun. Since activators make sequence-specific contacts with the DNA template at various positions upstream of the core promoter, it is also plausible that activators bound to unique surfaces of TFIID can influence specific structures of a promoter as the DNA traverses along TFIID resulting in distinct activator/promoter DNA structures (Liu, 2009).
Activator mapping results also complement and structurally extend the functional relevance of previous biochemical and immunomapping studies of TFIID. For example, label transfer studies show that the N-terminal activation domain of p53 contacts TAF6, confirming previous biochemical evidence showing that amino acids 1-42 of p53 contact TAF6/9. In support of this observation, the p53-IID 3D structure indicates that p53 contacts TFIID at lobes A and C where TAF6/9 are located as determined by EM immunomapping. In addition, previous studies have shown that both TBP and TAF1 can directly contact p53 in the absence of additional TFIID subunits. Interestingly, body-labeled p53 cross-linked to TAF1, TAF5, and weakly to TBP, thus extending the immunomapping studies that determined the locations of TBP and the N terminus of TAF1 at lobe C. Thus, EM activator mapping studies show a significant interface between p53 and specific TAFs located at lobes A and C of TFIID. Likewise, Sp1 label transfer results confirmed previous biochemical data showing a direct interaction between TAF4 and the N-terminal glutamine-rich domains of Sp1. In addition to TAF4, TAF6 was identified as weakly cross-linked to Sp1, suggesting that TAF6 may also be in the vicinity but perhaps more distal to the N terminus of Sp1. The largest TFIID subunit, TAF1, was cross-linked when body-labeled Sp1 was used. This result was not entirely unexpected, since previous studies found that TAF1 is required for Sp1-dependent transactivation, possibly through a direct interaction between TAF1 and Sp1 (Liu, 2009).
In comparison with p53 and Sp1, body-labeled c-Jun was shown to contact TAF1 and TAF6 in label transfer studies with no subunits contacting the N-terminal activation domain of c-Jun. This N-terminal activation domain of c-Jun may be structurally flexible or predominantly unstructured and is apparently positioned away from TFIID contacts. Indeed, successful structural studies of c-Jun thus far have been limited to the C-terminal leucine zipper DNA-binding region when bound to DNA. Previous biochemical assays have shown that the C-terminal basic leucine zipper DNA-binding region also contacts the N terminus of TAF1 (Liu, 2009).
It is worth noting that the extra density representing c-Jun and the other activator polypeptides in EM studies may not reflect the full-expected size of the activators. This is due to the presence of large unstructured regions in these proteins that are averaged out during structural analysis. As activators contain multiple molten globular domains that likely interact with different partners, one would expect a high degree of structural disorder in the domains that are not in direct contact with TFIID. Thus, the extra density associated with each activator determined from the single-particle reconstructions likely only represents minimally the most stably associated portion of activators bound to TFIID. This common situation would invariably lead to underrepresenting the actual size of the activator in a manner not unlike crystal structures of domains with flexible loops that become 'invisible' in the crystal structure (Liu, 2009).
Based on EM immunomapping, there are two copies of TAF6 within TFIID, wherein one copy resides in lobe A and another in lobe B. Collectively, the current studies suggest that two distinct activators (p53 and c-Jun) strongly contact the two different TAF6 subunits that are each located in different lobes of TFIID. It is unknown how p53 or c-Jun discriminates between TAF6 on lobe A versus B when binding to TFIID. In the future, it will be interesting to investigate if these two activators can bind to a single TFIID molecule simultaneously and decipher 3D structures of TFIID assemblies bound to select endogenous promoter DNA sequences in the presence and absence of distinct activators that are engaged in synergistic transcriptional activation (Liu, 2009).
It is of note that unlike the radical, diverse, and global structural changes observed with CRSP/Mediator complexes upon activator binding, TFIID largely retains its overall architecture when bound by three different activators. Interestingly, this study found that two of the activator/IID structures, p53-IID and Sp1-IID assemblies appear to be more constricted around the central cavity with narrower ChB-D and ChA-B channels, while the third structure, c-Jun-IID, remains most similar to free holo-TFIID. In particular, the p53-IID structure more closely resembles the closed conformational state of the previous cryo-TFIID structure. To test if p53-bound TFIID mimics the most closed conformational form of holo-TFIID, 3D reconstructions were performed using either the most closed or 'open' cryo-TFIID structures as an initial reference volume for refinement. Interestingly, it was found that both newly refined 3D structures generated from either the closed or open reference volume are fairly similar, with possibly a partial occupancy of p53 on lobe A. These findings suggest that the overall p53-TFIID structure tends to move toward the closed conformation with moderate movement at the outer tips of lobes A and B, even though p53-IID is predominantly observed in an intermediate average conformational form between the most closed and open forms. Perhaps factors contacting lobe A or C can induce certain coordinated movements within lobes that lead to a closed conformation of TFIID (Liu, 2009).
Although TFIID largely retains its prototypic global architecture upon activator binding, several common localized structural changes induced upon activator binding were observed in the 3D reconstruction. For example, a prominent and consistent induced extra density protrusion located in lobe D was observed when each of the three different activators binds TFIID. Given that all these activators are represented by distinct densities with unique sizes and shapes within the bound TFIID structure, and the fact that it has been demonstrated that they each can target different subunits within TFIID by a number of independent biochemical assays, it seems reasonable to assign 'unique and significant' extra densities located at distinct sites as representing the different bound activators. In contrast, the common similarly sized extra density seen at lobe D of each activator-IID structure most likely represents a conserved conformational change induced by these three different activators. Interestingly, this protrusion in lobe D resides distal to each of the activator-binding sites, suggesting that these three activators may potentially induce a long-range internal conformational change within TFIID. It would be intriguing to identify which TAF subunits are located at the tip of lobe D and eventually determine the function, if any, of this extended lobe in activator-induced transcription initiation. However, despite the potential significance of these structural changes induced by activators, it is premature to speculate regarding their functional importance (Liu, 2009).
Sp1 nuclear levels have been shown to directly correlate with the proliferative state of the cell. Changes in the abundance of Sp1 were studied in a rat pituitary cell line (GH4) whose growth rate is
regulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nM
EGF for at least 16 h show a 50% decrease in Sp1 binding to a GC-rich DNA sequence element present in the
gastrin promoter. The decrease in binding correlates with a decrease in cell proliferation, a loss of
nuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancer
element in transfection assays. Okadaic acid, a phosphatase inhibitor, is synergistic with the effect of
EGF on Sp1 protein levels, suggesting that the loss of Sp1 is mediated by phosphorylation events.
A 2-fold increase in orthophosphate-labeled Sp1 occurs with EGF treatment and
okadaic acid. Cycloheximide prevents the expected loss of Sp1 mediated by EGF and okadaic acid. This
suggests that the synthesis of a protease may mediate these events. This hypothesis was tested
directly by showing that the cysteine protease inhibitor leupeptin prevents Sp1 degradation. Sp1 has a domain with a high concentration of proline, glutamic acid, serine,
and threonine residues, as reported for a number of proteins with inducible rates of degradation.
Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade of
phosphorylation events that promotes Sp1 proteolysis, decreases Sp1 nuclear levels and decreases
cellular proliferation (Mortensen, 1997).
Muscle cell differentiation causes a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels.
A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drives transcriptional activity in
myoblasts, and differentiation causes a decrease in transcription. The fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced
transcription after muscle differentiation. Sp1 protein binds to the fragment
-102/-37 in the myoblast state but not in myotubes, and Sp1 is found to transactivate the GLUT1 promoter. Sp1 is drastically down-regulated during myogenesis. The forced over-expression of MyoD (Drosophila homolog: Nautilus) in
C3H10T1/2 cells mimics the effects observed during myogenesis: Sp1 down-regulation and reduced transcriptional activity of the
GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the
down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in
GLUT1 expression and glucose transport (Vinals, 1997).
Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. The mouse Sp3 gene has been disrupted by
homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced
defect in late tooth formation. Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. However, core binding factor 1 (Cbfa1), the transcription factor that is essential for bone formation, is
expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for
correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is
significantly diminished. These studies establish Sp3 as an essential transcription factor for late bone development (Gollner, 2001).
Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing transcription of cell cycle regulators, including cyclin D1. Evidence is provided that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, protein-DNA interactions have been identified within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which has been termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. The finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter (Hilton, 2003).
A missense mutation within the histone acetyltransferase (HAT) domain of the TATA binding protein-associated factor TAF1 induces ts13 cells to undergo a late G(1) arrest and decreases cyclin D1 transcription. TAF1 mutants (Delta844-850 and Delta848-850, from which amino acids 844 through 850 and 848 through 850 have been deleted, respectively) deficient in HAT activity are unable to complement the ts13 defect in cell proliferation and cyclin D1 transcription. Chromatin immunoprecipitation assays revealed that histone H3 acetylation is reduced at the cyclin D1 promoter but not the c-fos promoter upon inactivation of TAF1 in ts13 cells. The hypoacetylation of H3 at the cyclin D1 promoter is reversed by treatment with trichostatin A (TSA), a histone deacetylase inhibitor, or by expression of TAF1 proteins that retain HAT activity. Transcription of a chimeric promoter containing the Sp1 sites of cyclin D1 and c-fos core remain TAF1 dependent in ts13 cells. Treatment with TSA restores full activity to the cyclin D1-c-fos chimera at 39.5 degrees C. In vivo genomic footprinting experiments indicate that protein-DNA interactions at the Sp1 sites of the cyclin D1 promoter are compromised at 39.5 degrees C in ts13 cells. These data have led to a hypothesis that TAF1-dependent histone acetylation facilitates transcription factor binding to the Sp1 sites, thereby activating cyclin D1 transcription and ultimately G(1)-to-S-phase progression (Hilton, 2005).
Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological
functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis.
Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of
certain cancer cells. A novel class of synthetic retinoids has been identified with strong anti-cancer
cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an
electrophoretic mobility shift assay, the DNA binding activity of several transcription
factors was examined in T cells treated with apoptotic retinoids. The DNA binding activity of the
general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1
protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells
contain a protease activity that specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can
be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates
cleavage of Sp1. Inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests
that caspases are directly involved in this event. In fact, caspases 2 and 3 (see Drosophila Caspase 1) are activated in T cells after
treatment with apoptotic retinoids. The peptide inhibitors also block retinoid-induced apoptosis, as
well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells.
Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on
the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de
novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved,
triggering cell death in a transcriptional activation-independent, caspase-dependent manner (Piedrafita, 1997).
Continued: Evolutionary Homologs part 2/2
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