blistered/Serum response factor
Studies of mammalian SRF in cultured cells have focused on possible roles in mitogenesis, but additional evidence implcates SRF in regulation of the cytoskeleton. SRF targets include a number of cytoskeletal genes, and treatment of cells with agents that disrupt the cytoskeleton activates SRF-mediated transcription. Recent evidence has implicated SRF in signaling pathways involving the small GTPases, Rho, Rac and CDC42 that regulate cytoskeletal rearrangements. Perhaps Rho, Rac, and CDC42 activation of SRF stimulates expression of genes that reinforces the more direct effects of these small GTPases on cytoskeleton (Treisman, 1995, Chant, 1995, Guillemin, 1996 and references).
The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization,
transcription and transformation. Although many different potential RhoA effectors have been
identified, including two families of protein kinases, their roles in RhoA-regulated events remain
unclear. A genetic screen was used to identify mutations at positions 37-42 in the RhoA effector loop
that selectively disrupt effector binding, and these were used to investigate the role of RhoA effectors in the
formation of actin stress fibres, activation of transcription by serum response factor (SRF) and
transformation. Interaction with the ROCK kinase and at least one other unidentified effector is
required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of
RhoA-induced cytoskeletal changes, and does not correlate with binding to any of the effectors tested,
indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of
SRF, correlates with the activity of RhoA in transformation. The effector mutants should provide novel
approaches for the functional study of RhoA and isolation of effector molecules involved in specific
signalling processes (Sahai, 1998).
RhoA and two other Rho-family proteins, Cdc42 and Rac1, regulate Serum Response Factor (SRF) activation of the c-fos serum response element. This pathway acts independently of known MAPK pathways and is regulated by agents such as serum and LPA, acting via heterotrimeric G protein-coupled receptors.
Constitutively active forms of either of the small GTPases -- RhoA (RhoA.V14) or Cdc42 (Cdc42.V12) -- induces
expression of extrachromosomal SRF reporter genes in microinjection experiments, but only
Cdc42.V12 can efficiently activate a chromosomal template. Both SAPK/JNK-dependent or
-independent signals can cooperate with RhoA.V14 to activate chromosomal SRF reporters; it is
SAPK/JNK activation by Cdc42.V12 that allows SAPK/JNK to activate chromosomal templates. Cooperating
signals can be bypassed by deacetylase inhibitors. Three findings show that histone H4
hyperacetylation is one target for cooperating signals, although it alone is not sufficient: (1) Cdc42.V12,
but not RhoA.V14, induces H4 hyperacetylation; (2) cooperating signals use the same
SAPK/JNK-dependent or -independent pathways to induce H4 hyperacetylation, and (3) growth factor and
stress stimuli induce substantial H4 hyperacetylation, detectable in reporter gene chromatin. These data
establish a link between signal-regulated acetylation events and gene transcription. Thus, in isolation, the SRF-controlled extrachromosomal reporter gene is a target for only a subset of signals that can activate the chromsomal c-fos promoter. This is thought to reflect differences in chromatin structure associated with the two types of templates (Alberts, 1998).
A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec
non-receptor tyrosine kinase family. Tec and Bmx are found to activate serum response factor (SRF), in synergy with
constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The
SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the
kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and
Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where
thrombin's effect is mediated by G12/13 proteins. Expression of Galpha12 and Galpha13 stimulates the
autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in
Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which has been previously shown to activate kinase
activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3
does not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is
overexpressed. Thus, Tec/Bmx non-receptor tyrosine kinases are involved in regulation of
Rho and serum response factor by Galpha12/13 (Mao, 1998a).
Serum response factor (SRF) regulates transcription of many serum-inducible and muscle-specific genes. Using a functional screen, LIM kinase-1 has been identified as a potent activator of SRF. SRF activation by LIM kinase-1 is dependent on its ability to regulate actin treadmilling. LIM kinase activity is not essential for SRF activation by serum, but signals depend on alterations in actin dynamics. Studies with actin-binding drugs, the actin-specific C2 toxin, and actin overexpression demonstrate that G-actin level controls SRF. Regulation of actin dynamics is necessary for serum induction of a subset of SRF target genes, including vinculin, cytoskeletal actin, and srf itself, and also suffices for their activation. Actin treadmilling provides a convergence point for both serum- and LIM kinase-1-induced signaling to SRF (Sotiropoulos, 1999).
The small GTPase RhoA controls activity of serum response factor (SRF) by inducing changes in actin dynamics. In PC12 cells, activation of SRF after serum stimulation is RhoA dependent, requiring both actin polymerization and the Rho kinase (ROCK)-LIM kinase (LIMK)-cofilin signaling pathway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways (Geneste, 2002).
Serum response factor (SRF) regulates transcription of numerous muscle and growth factor-inducible genes. Because SRF is not muscle specific, it has been postulated to activate muscle genes by recruiting myogenic accessory factors. Using a bioinformatics-based screen for unknown cardiac-specific genes, a novel and highly potent transcription factor, named myocardin, was identified that is expressed in cardiac and smooth muscle cells. Myocardin belongs to the SAP domain family of nuclear proteins and activates cardiac muscle promoters by associating with SRF. Expression of a dominant negative mutant of myocardin in Xenopus embryos interferes with myocardial cell differentiation. Myocardin is the founding member of a class of muscle transcription factors and provides a mechanism whereby SRF can convey myogenic activity to cardiac muscle genes (Wang, 2001).
Myocardin is a SAP (SAF-A/B, Acinus, PIAS) domain transcription factor that associates with serum response factor (SRF) to potently enhance SRF-dependent transcription. Two myocardin-related transcription factors (MRTFs), A and B, are described that also interact with SRF and stimulate its transcriptional activity. Whereas myocardin is expressed specifically in cardiac and smooth muscle cells, MRTF-A and -B are expressed in numerous embryonic and adult tissues. In SRF-deficient embryonic stem cells, myocardin and MRTFs are unable to activate SRF-dependent reporter genes, confirming their dependence on SRF. Myocardin and MRTFs comprise a previously uncharacterized family of SRF cofactors with the potential to modulate SRF target genes in a wide range of tissues (Wang, 2002).
Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that activates serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, its role in serum induction of c-fos and other SRE-regulated genes was assessed with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). DN-MKL1 and RNAi was found to specifically block SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi requires the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduces the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, is nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia has a markedly increased ability to activate SRE reporter genes, suggesting that fusion protein activation of SRF target genes may contribute to leukemogenesis (Cen, 2003).
Rho GTPases regulate the transcription factor SRF via their ability to induce
actin polymerization. SRF activity responds to G actin, but the mechanism of
this has remained unclear. Rho-actin signaling is shown to regulate the
subcellular localization of the myocardin-related SRF coactivator MAL,
rearranged in t(1;22)(p13;q13) AML. The MAL-SRF interaction displays the
predicted properties of a Rho-regulated SRF cofactor. MAL is predominantly
cytoplasmic in serum-starved cells, but accumulates in the nucleus following
serum stimulation. Activation of the Rho-actin signaling pathway is necessary
and sufficient to promote MAL nuclear accumulation. MAL N-terminal sequences,
including two RPEL motifs, are required for the response to signaling, while
other regions mediate its nuclear export (or cytoplasmic retention) and nuclear
import. MAL associates with unpolymerized actin through its RPEL motifs.
Constitutively cytoplasmic MAL derivatives interfere with MAL redistribution and
Rho-actin signaling to SRF. MAL associates with several SRF target promoters
regulated via the Rho-actin pathway (Mirales, 2003).
Considered together, these data suggest a simple scheme for regulation of MAL
subcellular localization by Rho-actin signaling. MAL localization is
regulated in response to the level of either G actin itself or a G actin
subpopulation defined by interaction with actin binding proteins or
nucleotide-loading status. In the simplest model, G actin, perhaps acting with a
cofactor, would titrate MAL and thereby occlude nuclear import signals or create
a nuclear export signal; in either case, depletion of the G actin pool by
Rho-induced actin polymerization would result in MAL nuclear accumulation.
MAL nuclear accumulation can also be induced by actin binding
drugs, which directly interfere with actin-MAL association. The possibility cannot be excluded
that an additional positively acting G actin subpopulation exists which binds
MAL in such a way as to promote its nuclear accumulation.
The data do not address the mechanism of MAL downregulation: it is likely that
additional regulatory mechanisms must operate, since MAL remains nuclear even
after the shutdown of target gene expression (Mirales, 2003).
Serum stimulation also induces MAL C-terminal phosphorylation via both Rho- and
MEK-ERK dependent signal pathways. The finding that nuclear accumulation of MAL
mutants lacking the C-terminal sequences is still dependent on Rho signaling,
however, suggests that phosphorylation is not a prerequisite for serum-induced
MAL nuclear accumulation and SRF activation. Consistent with this idea,
expression of activating actin mutants can
induce nuclear accumulation of MAL without its concomitant phosphorylation.
Nevertheless, the possibility cannot be excluded that
regulated phosphorylation of MAL may control its nuclear accumulation under
circumstances in which signaling does not regulate actin dynamics. It also
remains possible that phosphorylation regulates nuclear export, cytoplasmic
retention, or even transcriptional activation (Mirales, 2003).
Serum response factor (SRF) is required for the expression of a wide variety of muscle-specific genes that are expressed upon differentiation and is thus required for both striated and smooth muscle differentiation in addition to its role in regulating growth factor-inducible genes. A heart and smooth muscle-specific SRF co-activator, myocardin, has been shown to be required for cardiac development and smooth muscle differentiation. However, no such co-factors of SRF have been identified in the skeletal myogenic differentiation program. Myocardin and the related transcription factor megakaryoblastic leukemia-1 (MKL1/MAL/MRTF-A) can strongly potentiate the activity of SRF. The third member of the myocardin/MKL family in humans, MKL2, has been cloned. MKL2 binds to and activates SRF similarly to myocardin and MKL1. To determine the role of these factors in skeletal myogenic differentiation a dominant negative MKL2 was used to show that the MKL family of proteins is required for skeletal myogenic differentiation. Expression of the dominant negative protein in C2C12 skeletal myoblasts blocks the differentiation-induced expression of the SRF target genes skeletal alpha-actin and alpha-myosin heavy chain and blocks differentiation of the myoblasts to myotubes in vitro. C2C12 cells express both MKL1 and MKL2, but not myocardin, implicating MKL1 and/or MKL2 in the requirement for skeletal myogenic differentiation. MKL1 is predominantly cytoplasmic in C2C12 cells, with a small amount in the nucleus, however, no movement of MKL1 to the nucleus was observed upon differentiation (Selvaraj, 2003).
The SAP domain transcription factor myocardin plays a critical role in the transcriptional program regulating smooth muscle cell differentiation. Myocardin is shown to physically associate with megakaryoblastic leukemia factor-1 (MKL1), and this report describes the function of MKL1 in smooth muscle cells (SMCs). The MKL1 gene is expressed in most human tissues and myocardin and MKL are co-expressed in SMCs. MKL1 and myocardin physically associate via conserved leucine zipper domains. MKL1 has been shown to regulate the transcriptional activity of SRF in fibroblasts, via nuclear translocation in response to serum, RhoA, and actin treadmilling. Overexpression of MKL1 transactivates serum response factor (SRF)-dependent SMC-restricted transcriptional regulatory elements including the SM22alpha promoter, smooth muscle myosin heavy chain promoter/enhancer, and SM-alpha-actin promoter/enhancer in non-SMCs. Moreover, forced expression of MKL1 and SRF in undifferentiated SRF(-/-) embryonic stem cells activates multiple endogenous SMC-restricted genes at levels equivalent to, or exceeding, myocardin. Forced expression of a dominant-negative MKL1 mutant reduces myocardin-induced activation of the SMC-specific SM22alpha promoter. In NIH3T3 fibroblasts MKL1 localizes to the cytoplasm and translocates to the nucleus in response to serum stimulation, actin treadmilling, and RhoA signaling. In contrast, in SMCs MKL1 is observed exclusively in the nucleus regardless of serum conditions or RhoA signaling. However, when actin polymerization is disrupted MKL1 translocates from the nucleus to the cytoplasm in SMCs. Together, these data were consistent with a model wherein MKL1 transduces signals from the cytoskeleton to the nucleus in SMCs and regulates SRF-dependent SMC differentiation autonomously or in concert with myocardin (Du, 2004).
Nuclear accumulation of the serum response factor coactivator MAL/MKL1
is controlled by its interaction with G-actin, which results in its retention in
the cytoplasm in cells with low Rho activity. Actin
mutants have been identified whose expression promotes MAL nuclear accumulation via an unknown
mechanism. Actin interacts directly with MAL in vitro with
high affinity. A further activating mutation, G15S, has been identified that stabilises
F-actin, as do the activating actins S14C and V159N. The three mutants share
several biochemical properties, but can be distinguished by their ability to
bind cofilin, ATP and MAL. MAL interaction with actin S14C is essentially
undetectable, and that with actin V159N is weakened. In contrast, actin G15S
interacts more strongly with MAL than the wild-type protein. Strikingly, the
nuclear accumulation of MAL induced by overexpression of actin S14C is
substantially dependent on Rho activity and actin treadmilling, while that
induced by actin G15S expression is not. A model is proposed in which actin G15S
acts directly to promote MAL nuclear entry (Posern, 2004).
This paper studies the connection between activating actin mutants and SRF activation in the light of the identification of MAL as an actin-regulated SRF coactivator. Wild-type actin binds the N-terminal RPEL domain of MAL directly in vitro, and a new activating actin mutant, actin G15S, was identified in a two-hybrid screen with this domain. All three activating actin mutants, actins G15S, S14C and V159N, share the ability to stabilise F-actin. They all exhibit a decreased ability to bind the C-terminal half of gelsolin, and an enhanced affinity for profilin, suggesting that their structure mimics that of ATP-actin. However, they can also be distinguished biochemically by their ability to interact with nucleotide, cofilin and MAL. Although actin G15S binds MAL more strongly than wild-type actin, the S14C mutation greatly reduces the actin-MAL interaction. Strikingly, the ability of actin S14C to induce MAL nuclear accumulation is strongly dependent on the actin treadmilling cycle and basal Rho activity, while G15S- and V159N-induced MAL translocation is not. It is proposed that whereas actin S14C releases MAL from the inhibitory effect of wild-type G-actin by diluting the endogenous G-actin pool, actin G15S (and probably V159N) may act directly to induce MAL nuclear translocation (Posern, 2004).
Myocardin (Drosophila homolog: Myocardin-related transcription factor) is a cardiac- and smooth muscle-specific cofactor for the ubiquitous transcription factor serum response factor (SRF). Using gain-of-function approaches in the Xenopus embryo, myocardin was shown to be sufficient to activate transcription of a wide range of cardiac and smooth muscle differentiation markers in non-muscle cell types. For the myosin light chain 2 gene (MLC2), myocardin cooperates with the zinc-finger transcription factor Gata4 to activate expression. Inhibition of myocardin activity in Xenopus embryos using morpholino knockdown methods results in inhibition of cardiac development and the absence of expression of cardiac differentiation markers and severe disruption of cardiac morphological processes. It is concluded that myocardin is an essential component of the regulatory pathway for myocardial differentiation (Small, 2005).
Myocardin and the myocardin-related transcription factors (MRTFs) MRTF-A and MRTF-B are coactivators for serum response factor (SRF), which regulates genes involved in cell proliferation, migration, cytoskeletal dynamics, and myogenesis. MRTF-A has been shown to translocate to the nucleus and activate SRF in response to Rho signaling and actin polymerization. A muscle-specific actin-binding protein named striated muscle activator of Rho signaling (STARS) also activates SRF through a Rho-dependent mechanism. STARS activates SRF by inducing the nuclear translocation of MRTFs. The STARS-dependent nuclear import of MRTFs requires RhoA and actin polymerization, and the actin-binding domain of STARS is necessary and sufficient for this activity. A knockdown of endogenous STARS expression by using small interfering RNA significantly reduces SRF activity in differentiated C2C12 skeletal muscle cells and cardiac myocytes. The ability of STARS to promote the nuclear localization of MRTFs and SRF-mediated transcription provides a potential muscle-specific mechanism for linking changes in actin dynamics and sarcomere structure with striated muscle gene expression (Kuwahara, 2005).
Serum response element binding protein (SRE BP) is a novel binding factor present in
nuclear extracts of avian and NIH 3T3 fibroblasts that specifically bind to the cfos
SRE within a region overlapping and immediately 3' to the CArG box. Binding of both Serum response factor (SRF) and SRE BP is necessary for maximal serum induction of the SRE. Homodimers and heterodimers of p35C/EBP (a transactivator) and
p20C/EBP (a repressor) contribute to the SRE BP complex in NIH 3T3 cells. Transactivation of the SRE by p35C/EBP is dependent on SRF binding but not ternary complex factor (TCF) formation. Both p35C/EBP and p20C/EBP bind to SRF in vitro via a carboxy-terminal domain that probably does not include the leucine zipper. Moreover, SRE mutants that retain responsiveness to the TCF-independent signaling pathway bind SRE BP in vitro with affinities that are nearly identical to that of the wild-type SRE, whereas another mutant, which is not responsive to the TCF-independent pathway, have a nearly 10-fold lower affinity for SRE BP. It is proposed that C/EBP may play a role in conjunction with SRF in the TCF-independent
signaling pathway for SRE activation (Sealy, 1997).
The human paired class homeodomain protein Phox1 (which has no known Drosophila homolog) can impart serum-responsive transcriptional activity to the
c-fos serum response element (SRE) by interacting with serum response factor (SRF). This activity is
shared with other paired class homeodomains but not with more distantly related homeodomains. To
understand the mechanism of Phox1 action at the SRE and the basis for the selective activity of
Paired class homeodomains in this context, a detailed mutagenesis was performed of the Phox1
homeodomain. Homeodomain amino acid residues that contact the major groove of the DNA are
required for SRE activation in vivo. In contrast, substitution of a lysine residue in the N-terminal arm of the Phox1
homeodomain appears to abolish DNA binding without affecting activity in vivo. Certain substitutions
on the exposed surfaces of helices 1 and 2, not required for DNA binding, abolish activity in vivo,
suggesting that these surfaces contact an accessory protein(s) required for this activity. Transfer of a single amino acid residue from the surface of Phox1 helix 1 to the corresponding
position in the distantly related Deformed (Dfd) homeodomain imparts to Dfd the ability to activate the
SRE in vivo. It is proposed that Phox1 interacts with one or more factors at the SRE, in addition to SRF,
and that the specificity of this interaction is determined by residues on the surfaces of helices 1 and 2 (Simon, 1997).
The human homeodomain protein Phox1 interacts functionally with Serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. This study reports the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. This protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. It is proposed that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery (Grueneberg, 1997).
Endothelins are a family of biologically active peptides that are critical for development and function of neural crest-derived and
cardiovascular cells. These effects are mediated by two G-protein-coupled receptors and involve transcriptional regulation of
growth-responsive and/or tissue-specific genes. The cardiac ANF promoter, which represents the best-studied tissue-specific endothelin target, has been used to elucidate the nuclear pathways responsible for the transcriptional effects of endothelins. Cardiac-specific response to endothelin 1 (ET-1) requires the combined action of the serum response factor (SRF)
and the tissue-restricted GATA proteins that bind over their adjacent sites, within a 30-bp ET-1 response element. SRF and GATA proteins
form a novel ternary complex reminiscent of the well-characterized SRF-ternary complex factor interaction required for transcriptional induction of c-fos in response to growth factors. In transient cotransfections, GATA factors and SRF synergistically activate atrial natriuretic factor and other ET-1-inducible
promoters that contain both GATA and SRF binding sites. Thus, GATA factors may represent a new class of tissue-specific SRF accessory factors that
account for muscle- and other cell-specific SRF actions (Morin, 2001).
SRF, initially isolated as the nuclear protein that mediates
transcriptional response of c-fos and other immediate-early
genes to growth factors, has been one of the most extensively
characterized transcription factors. It is now well established that many SRF-dependent responses to growth factor stimulation are mediated by an
SRF-containing ternary complex in which the TCF is the target of
several MAPK cascades. At least three different but related TCFs have been identified; functional as well as structural analyses of the TCF-SRF-DNA ternary complex suggest that TCFs act as growth-regulated SRF cofactors.
Unexpectedly, while mutations that abolish TCF binding render the
c-fos promoter unresponsive to some growth factors, they do
not abolish serum regulation or endothelin stimulation. This has led to the speculation that an unidentified SRF cofactor that would interact with the DNA-binding SRF domain and form a ternary complex with SRF and DNA must exist. These results suggest that GATA factors may fulfill these criteria. Indeed, GATA-4 and -6 interact with the DNA-binding domain of SRF and form a stable ternary complex, as evidenced by gel shift analysis and supported by molecular modeling. Remarkably, it was found that the well-studied c-fos SRE contains two inverted GATA motifs flanking the SRF binding sequences that bind recombinant GATA factors, albeit with lower affinity than the ANF GATA sites.
Moreover, the c-fos promoter as well as a c-fos
SRE heterologous promoter are synergistically activated by SRF and
GATA factors in many cell types. Whether a GATA-SRF ternary complex can substitute for the TCF-SRF complex over the c-fos promoter and mediate cell-specific serum or growth-differentiation responses in GATA-expressing cells deserves to be investigated (Morin, 2001).
Activin/Nodal signaling is essential for germ-layer formation and axial patterning during embryogenesis. Recent evidence has demonstrated that the intra- or extracellular inhibition of this signaling is crucial for ectoderm specification and correct positioning of mesoderm and endoderm. This study analyzed the function of Xenopus serum response factor (XSRF) in establishing germ layers during early development. XSRF transcripts are restricted to the animal pole ectoderm in Xenopus early embryos. Ectopic expression of XSRF RNA suppresses mesoderm induction, both in the marginal zone in vivo and caused by Activin/Nodal signals in animal caps. Dominant-negative mutant or antisense morpholino oligonucleotide-mediated inhibition of XSRF function expands the expression of mesendodermal genes toward the ectodermal territory and enhances the inducing activity of the Activin signal. SRF interacts with Smad2 and FAST-1, and inhibits the formation of the Smad2-FAST-1 complex induced by Activin. These results suggest that XSRF might act to ensure proper mesoderm induction in the appropriate region by inhibiting the mesoderm-inducing signals during early embryogenesis (Yun, 2007; full text of article).
SRF acts as a Smad2-binding partner to inhibit Activin/Nodal-dependent transcription. Recent evidence points to several mechanisms by which interference with Smad transcriptional complexes negatively regulates TGF-β signaling. For instance, the oncoprotein Ski, a transcriptional co-repressor, competes with R-Smads for association with Smad4, disrupting the formation of a functional complex between Smad4 and R-Smads. Moreover, Ski can also repress Smads directly by recruiting the transcriptional repressor N-CoR as well as the histone deacetylase complex (HDAC). DRAP1 interacts with FAST-1, thereby preventing FAST-Smad2-Smad4 complex from binding to its cognate DNA targets. In addition, inhibitory Smads (Smad6 and Smad7) compete with R-Smads for binding to activated type-I receptors and thus inhibit the phosphorylation of R-Smads. The data show that SRF precludes the association of Smad2 and FAST-1 induced by Activin signal. This suggests that SRF could function to impede Smad2-FAST complex-mediated transcription in Activin/Nodal signaling. Supporting this, gain-of-function phenotypes of SRF are similar to those of maternal FAST-depleted embryos and can be rescued by coexpression of an activated mutant of FAST-1 (FAST-VP16A). The possibility cannot be excluded that SRF could also affect FAST-independent transcription, since FoxH1 depletion in animal caps has no effect on the induction of Nodal target genes in response to Xnr1 or Activin ligands, whereas overexpression of XSRF significantly inhibits the same response. Given that Smad2 binds to SRF via its MH2 domain, which associates with the general transcriptional co-activators p300 and CAATT-binding factor (CBF), SRF may repress the Smad2-mediated transcription of various genes in a cell context-dependent manner by preventing the interaction of the MH2 domain and these co-activators. By contrast, a recent study shows that SRF associates with Smad3 and activates TGF-β1-dependent transcription during myofibroblast differentiation. In contrast, the general mechanism by which SRF regulates gene transcription is known to involve cooperation with the ternary complex factors (TCF), which are phosphorylated and activated by MAP kinase cascades. In Xenopus, SRF was shown, together with the TCF-type Ets protein Elk-1, to regulate the transcription of Xegr-1, an organizer-specific gene, downstream of the FGF-initiated MAP kinase pathway. Interestingly, TGF-β receptors can activate MAP kinase signaling pathways. These activated MAP kinase cascades inhibit or enhance Smad activity by phosphorylating it, depending on the cell signaling context; but, in some cases, they regulate Smad-independent transcription. It will be interesting to examine whether the TCF- and the Smad-dependent SRF regulation of gene transcription involve distinct signaling cascades or whether both of them could be controlled via MAPK pathways by TGF-β signaling. In addition, it remains to be investigated in more detail how SRF could regulate gene expression in a positive or negative fashion depending on its transcription-factor binding-partners (Yun, 2007).
Krox-20, originally identified as a member of 'immediate-early' genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. A genomic sequence of Xenopus Krox-20 (XKrox-20) has been cloned and a promoter element in the flanking sequence has been studied along with associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, it has been shown that the 5' flanking sequence is sufficient to induce luciferase activities when the reporter construct is injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5' flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box (sequence of the form CC (A/T)6 GG) and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (-72 to -51 from the transcription initiation site), both of which are essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bind to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts are serum response factor (SRF) and a member of CRE binding protein (CREB) family, proteins that bind the CArG-box and CRE, respectively. They appear to assemble on the minimal promoter element to produce a novel ternary complex. When mRNA of a dominant-negative version of Xenopus SRF (XSRFdeltaC) is injected into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity is strongly suppressed. Suppression by XSRFdeltaC is counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element (Watanabe, 2005).
Serum response factor (SRF) is a MADS box transcription factor that has been shown to be important in
the regulation of a variety of muscle-specific genes. SRF is a major component of multiple cis/trans interactions found along the smooth muscle gamma-actin (SMGA) promoter. The role of SRF was examined in the regulation of the SMGA gene in the developing gizzard. EMSA analyses, using nuclear extracts derived from gizzards at various stages in development, shows that the SRF-containing complexes are not present early in gizzard smooth muscle development, but appear as development progresses. Using Western and Northern blot analyses, an increase in SRF protein and mRNA levels is observed during gizzard development, with a large increase just preceding an increase in SMGA expression. Thus, changes in SRF DNA-binding activity are paralleled with increased SRF gene expression. Immunohistochemical analyses demonstrate a
correspondence of SRF and SMGA expression in differentiating visceral smooth muscle cells (SMCs)
during gizzard tissue development. This correspondence of SRF and SMGA expression is also
observed in cultured smooth muscle mesenchyme, induced to express differentiated gene products in
vitro. In gene transfer experiments with SMGA promoter-luciferase reporter gene constructs four- to fivefold stronger SMGA promoter activity is observed in differentiated SMCs relative to replicating visceral smooth muscle cells. The dominant negative SRF mutant protein is able to specifically inhibit transcription of the SMGA promoter in visceral smooth muscle, directly linking SRF with the control of SMGA gene expression. Taken together, these data suggest that SRF plays a prominent role in the developmental regulation of the SMGA gene (Browning, 1998).
The minimal muscle-specific dystrophin promoter contains the consensus sequence
CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or
muscle-specific promoters. The serum response factor, which mediates the
transcriptional activation of the c-fos gene in response to serum stimulation, can bind
to different CArG box elements, suggesting that it could be involved in
muscle-constitutive transcription. SRF binds to the dystrophin
promoter and regulates its muscle-specific transcription. An
altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of
a dystrophin promoter with a mutation in its CArG box element. The
muscle-constitutive transcription of the dystrophin promoter also requires the sequence
GAAACC immediately downstream of the CArG box. This sequence is recognized by
a novel DNA bending factor, named Dystrophin promoter-bending factor
(DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and
the promoter activity in muscle cells. Its replacement with a p62/ternary complex
factor binding site changes the promoter specificity from muscle constitutive to serum
responsive. These results show that on the dystrophin promoter the transcriptional
activation induced by SRF requires the DNA bending induced by DPBF. The bending,
next to the CArG box, could promote interactions between SRF and other proteins in
the transcriptional complex (Galvagni, 1997).
The SM22alpha promoter has been used as a model system to define the molecular mechanisms that
regulate smooth muscle cell (SMC) specific gene expression during mammalian development. The
SM22alpha gene is expressed exclusively in vascular and visceral SMCs during postnatal development
and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22alpha
promoter in transgenic mice reveals that 280 bp of 5' flanking sequence is sufficient to restrict
expression of the lacZ reporter gene to arterial SMCs and the myotomal component of the somites.
DNase I footprint and electrophoretic mobility shift analyses reveal that the SM22alpha promoter
contains six nuclear protein binding sites (designated smooth muscle elements [SMEs] -1 to -6,
respectively); two of these (SME-1 and SME-4) bind serum response factor (SRF). Mutational
analyses demonstrates that a two-nucleotide substitution that selectively eliminates SRF binding to
SME-4 decreases SM22alpha promoter activity in arterial SMCs by approximately 90%. Mutations that abolish binding of SRF to SME-1 and SME-4, or mutations that eliminate each SME-3
binding activity totally abolish SM22alpha promoter activity in the arterial SMCs and somites of
transgenic mice. A multimerized copy of SME-4 (bp -190 to -110) when
linked to the minimal SM22alpha promoter (bp -90 to +41) is necessary and sufficient to direct
high-level transcription in an SMC lineage-restricted fashion. Taken together, these data demonstrate
that distinct transcriptional regulatory programs control SM22alpha gene expression in arterial versus
visceral SMCs. These data are consistent with a model in which combinatorial interactions
between SRF and other transcription factors that bind to SME-4 (as well as those that bind directly to SRF)
activate transcription of the SM22alpha gene in arterial SMCs (Kim, 1997).
Recent evidence indicates that phosphatidylinositol 3-kinase (PI3K) is a central regulator of mitosis, apoptosis and oncogenesis.
Nevertheless, the mechanisms by which PI3K regulates proliferation are not well characterized. Mitogens stimulate entry into the cell
cycle by inducing the expression of immediate early genes (IEGs) that in turn trigger the expression of G1 cyclins. A
novel PI3K- regulated transcriptional cascade is described that is critical for mitogen regulation of the IEG, c-fos. PI3K activates gene
expression by transactivating SRF-dependent transcription, independent of the previously described Rho and ETS TCF pathways.
PI3K-stimulated cell cycle progression requires transactivation of SRF and expression of dominant- negative PI3K blocks
mitogen-stimulated cell cycle progression. Furthermore, dominant-interfering SRF mutants attenuate mitogen-stimulated cell cycle progression, but are without effect
on MEK-stimulated cell cycle entry. Moreover, expression of constitutively active SRF is sufficient for cell cycle entry. Thus, a novel SRF-dependent
mitogenic cascade is described that is critical for PI3K- and growth factor-mediated cell cycle progression (Poser, 2000).
What are the mitogenic targets of PI3K-stimulated gene expression?
Cyclin D1 transcription is induced by mitogens and cyclin D1 is thought to play a key role in mitogen-stimulated G1 progression. Recent evidence indicates that mitogens induce the expression and transcription of cyclin D1 in some cells via PI3K
signaling. Interestingly, growth factor-induced cyclin D1 transcription is blocked by expression
of dominant-negative PI3K or SRF. Because the cyclin D1 promoter does not contain an SRE, the requirement for SRF for cyclin D1 transcription is the result of the
indirect expression of SRF-regulated IEG transcription factors. For example, fibroblasts deficient for the SRF-regulated IEGs, c-fos and FosB, have a defect in
mitogen-induced proliferation and cyclin D1 transcription. Taken together, these results suggest that a mitotic target of PI3K-stimulated
SRF-mediated transcription is cyclin D1, which in turn mediates repression of Rb and activation of E2F (Poser, 2000).
Gradients of signalling and transcription factors govern many aspects of embryogenesis, highlighting the need for spatiotemporal control of regulatory protein levels. MicroRNAs are phylogenetically conserved small RNAs that regulate the translation of target messenger RNAs, providing a mechanism for protein dose regulation. Mammalian microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells. The miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2. Correspondingly, excess miR-1 in the developing heart leads to a decreased pool of proliferating ventricular cardiomyocytes. Using a new algorithm for microRNA target identification that incorporates features of RNA structure and target accessibility, Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is shown to be a target of miR-1. This work suggests that miR-1 genes titrate the effects of critical cardiac regulatory proteins to control the balance between differentiation and proliferation during cardiogenesis (Zhao, 2005).
The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to
extracellular stimuli, e.g. growth and differentiation signals. Complexes between DNA and protein, that contain SRF or its homologs, function as nuclear targets of the
Ras/MAPK signaling network, thereby directing gene activities associated with processes as diverse as pheromone signaling, cell-cycle progression
(transitions G0-G1 and G2-M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied
exclusively in cultured cells. To study SRF function in a multicellular organism, a Srf null allele was generated in mice. SRF-deficient embryos (Srf -/-)
have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive
streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp-2/4 and Shh. Activation of the SRF-regulated
immediate early genes Egr-1 and c-fos, as well as the alpha-Actin gene, is severely impaired. This study identifies SRF as a new and essential regulator of
mammalian mesoderm formation. It is therefore suggested that in mammals Ras/MAPK signaling contributes to mesoderm induction, as is the case in
amphibia (Arsenian, 1998).
Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of
the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix.
Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including
SMgammaactin, SM22gamma, calponin, SM(gama)actin or SM-myosin heavy chain detectable by RT-PCR
or by immunostaining. To identify factors required for coronary smooth muscle differentiation,
proepicardial cells from Hamburger-Hamilton stage-17 quail embryos were excised and examined ex vivo.
Proepicardial cells initially form an epithelial colony that is uniformly positive for cytokeratin, an
epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers are undetectable,
indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours,
cytokeratin-positive cells become SM(alpha)actin-positive. Moreover, serum response factor, undetectable in
freshly isolated proepicardial cells, become strongly expressed in virtually all epicardial cells. By 72 hours, a
subset of epicardial cells exhibits a rearrangement of cytoskeletal actin, focal adhesion formation and
acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22(alpha)
and SM(gama)actin are expressed. By 5-10 days, SM-myosin heavy chain mRNA is found, by which
time nearly all cells have become mesenchymal. RT-PCR shows that large increases in serum response
factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative
serum response factor constructs prevent the appearance of calponin-, SM22(alpha)- and
SM(gama)actin-positive cells. By contrast, dominant-negative serum response factor does not block
mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results
indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires
transcriptionally active serum response factor (Landerholm, 1999).
The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf-/- embryonic stem (ES) cells, it has been demonstrated that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), beta1-integrin, talin, zyxin, and vinculin are downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf-/- ES cells are accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescues F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf-/- ES cells, in contrast, strongly induces expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, the importance of SRF for the formation has been demonstrated of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. These findings suggest an involvement of SRF in cell migratory processes in multicellular organisms (Schratt, 2002).
The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. Ezh2 expression is developmentally regulated in the myotome compartment of mouse somites and its down-regulation coincides with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibits muscle differentiation, and this property is conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 is associated with the transcriptional regulator YY1. Both Ezh2 and YY1 are detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes: their presence correlates with methylation of K27 of histone H3. YY1 is required for Ezh2 binding because RNA interference of YY1 abrogates chromatin recruitment of Ezh2 and prevents H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociate from muscle loci, H3-K27 becomes hypomethylated and MyoD and SRF are recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation (Cartetti, 2004).
These results indicate that Ezh2 is recruited at the chromatin of selected muscle regulatory regions by the transcriptional regulator YY1. Both can be coimmunoprecipitated from myoblast and not myotube cell extracts, and the proteins colocalize at the same muscle chromatin regions in a developmentally regulated manner. The interaction of endogenous YY1 and Ezh2 is likely to be mediated by the PcG EED protein because recombinant YY1 and Ezh2 do not directly associate. Previous reports have demonstrated a negative role for YY1 in regulating muscle gene expression through interaction with distinct nucleotides within the CarG-box [CC(A+T-rich)6GG], one of the DNA elements required for muscle-specific gene transcription. Transcriptional activation coincides with replacement of YY1 by the serum response factor (SRF), whose interaction with the CarG-box is required for muscle-specific transcription to proceed. These data suggest a two-step activation model of muscle gene expression. In the repressed state, YY1 recruits a complex containing both Ezh2 and HDAC1 that silences transcription through histone methylation (H3-K27) and deacetylation. Transcriptional activation entails the initial removal of the YY1-Ezh2-HDAC1 repressive complex and subsequent recruitment of the activators SRF (which replaces YY1) and the MyoD family of transcription factors and associated acetyltransferases. Since YY1 binding tolerates a substantial nucleotide heterogeneity in its DNA recognition sites, muscle and non-muscle-specific CarG-less regulatory regions may be also occupied and regulated in a similar manner. In contrast, Ezh2 does not appear to promiscuously regulate expression of all muscle-specific genes as indicated by the transient coexpression of Ezh2 and myogenin in the myotome of developing embryos and lack of Ezh2 recruitment and H3-K27 methylation at the myogenin promoter. Distinct histone methyltransferases and deacetylases have been shown to modify histones at the myogenin promoter (Cartetti, 2004 and references therein).
Whereas significant insight exists as to how LTP-related changes can contribute to the formation of long-term memory, little is known about the role of hippocampal LTD-like changes in learning and memory storage. This study describes a mouse lacking the transcription factor SRF in the adult forebrain. This mouse could not acquire a hippocampus-based immediate memory for a novel context even across a few minute timespan, which led to a profound but selective deficit in explicit spatial memory. These animals were also impaired in the induction of LTD, including LTD triggered by a cholinergic agonist. Moreover, genes regulating two processes essential for LTD - calcium release from intracellular stores and phosphatase activation - were abnormally expressed in knockouts. These findings suggest that for the hippocampus to form associative spatial memories through LTP-like processes, it must first undergo learning of the context per se through exploration and the learning of familiarity, which requires LTD-like processes (Etkin, 2006).
Lack of SRF beginning in late gestation results in a severly hypoplastic hippocampus, evident on Nissl staining, and abnormal mossy fibers. These animals display phenotypic abnormalities by postnatal day 2, are much smaller than their littermates, and die by postnatal day 21. By deleting SRF only in adult mice, nervous system development was not impaired; this was verified in immunohistochemical analysis of hippocampi from SRF knockout mice (Etkin, 2006).
Previously published studies on SRF have focused on its role in neuronal activity-regulated gene transcription, which contributes to the late phase of LTP. By contrast, a deficit in LTD was observed beginning early during plasticity induction, suggesting that basal gene expression changes in knockout mice are more likely to account for the phenotype than stimulus-induced gene expression changes. Basal downregulation of the well-described SRF target genes zif268 and β-actin was observed. In addition, the expression of two major ryanodine receptors (RyR1 and 3) and one major inositol triphosphate receptor (IP3R1) were decreased in knockout brains. These receptors are important for the regulation of calcium release from intracellular stores (Etkin, 2006).
Using a combination of bath application and postsynaptic (CA1) intracellular injection experiments, several previous studies have shown that release of calcium from intracellular stores is essential for electrically induced LTD. Moreover, these studies found that ryanodine-regulated calcium release was required presynaptically, while IP3R1-regulated calcium release was required postsynaptically. These earlier experiments were extended by showing that depletion of intracellular calcium stores with CPA disrupts carbachol-induced LTD (Etkin, 2006).
One important function for calcium release in LTD is the activation of protein phosphatases, such as PP1. A roughly 2-fold increase was found in the expression of inhibitor-1, an endogenous inhibitor of PP1. Also downregulation was found of the inhibitor-1 regulators p35 and cdk5, which together suggest that knockouts have decreased PP1 activity, a function required for LTD (Etkin, 2006).
It has been found that postsynaptic (CA1) injection of the inhibitor-1 peptide blocks electrically induced LTD. Interestingly, in the limited electrophysiological and behavioral experiments reported, mice lacking p35 display an LTD deficit, no LTP deficit, and a learning curve in the water maze similar to SRF knockouts. In additional experiments, it was found that okadaic acid, which inhibits PP1 and PP2A, diminishes carbachol-induced LTD (Etkin, 2006).
One result of phosphatase activation is the clathrin-dependent endocytosis of AMPA receptors during LTD. It was also found that clathrin heavy chain was downregulated in knockouts, suggesting that clathrin-mediated endocytosis may also be affected in these mice. In summary, the basal gene expression and electrophysiological data suggest that decreased release of calcium from intracellular stores and decreased PP1 activation may together largely account for the LTD deficits observed in SRF knockout mice (Etkin, 2006).
When and how do LTD-like changes occur in vivo, and how is this relevant to the formation of an immediate memory of a novel environment? A portion of the population of hippocampal neurons (i.e., visually responsive neurons) fire robustly to aspects of a novel environment. Upon repeated exposure to the same stimuli, perhaps facilitated by the large release of acetylcholine, these neurons then reduce or eliminate their firing. Exposure to a novel environment led to an activity-dependent and long-lasting depression of synaptic transmission, as assessed by applying test pulses (which themselves do not alter synaptic efficacy) to the Schaffer collateral pathway in vivo. Similarly, a very rapid and robust LTD was observed during carbachol stimulation in response to test pulse stimulation. It is tempting to conclude that the novelty-induced LTD observed in vivo is mediated by the cholinergic modification of synaptic transmission in stimulated neurons, which were tapped into in vitro. The activity-dependence of novelty-induced and carbachol-induced LTD also illustrates how response decreases can be achieved specifically in those neurons responsive to aspects of the environment, since LTD-like changes will occur only if a neuron is active, even if at a low rate (Etkin, 2006).
LTD likely does not occur in vivo as a consequence of 15 min of continued 1 Hz stimulation. Very few, but well-timed, spikes can induce LTD if timed to coincide with the trough of hippocampal theta frequency oscillations, a rhythm commonly observed in vivo as animals explore their environment. Theta frequency oscillations depend on septal cholinergic innervation of the hippocampus, thereby providing another potential link between novelty, the increased release of acetylcholine, and the rapid induction of LTD in the hippocampus. Consistent with the rapid decrease in neural response in the hippocampus to repeated presentations of a novel stimulus, LTD deficits were observed in knockouts very early during induction of LTD (Etkin, 2006).
This study therefore suggests that LTD in the hippocampus is not merely a negative counterpart to LTP, acting as a restraint to memory storage, but provides a unique positive learning mechanism in its own right. Because those neurons undergoing LTD-like changes in a novel environment are responsive to sensory aspects of the context, they would also be candidate neurons for undergoing subsequent LTP-like changes during the formation of an explicit associative memory related to that context. By first undergoing LTD, the subset of neurons recruited for subsequent LTP would benefit from improved signal-to-noise. It is therefore suggested that LTD mediates a function in learning and memory that is distinct from that related to LTP, but that ultimately these two processes interact and affect each other. For the animal to encode complex spatial associative memories in a novel environment, such as for navigation using spatial cues, the animal must first encode that it has encountered the novel environment per se. This first step involves a distinct mnemonic process mediated by LTD-like changes and is evident behaviorally by habituation of exploration (Etkin, 2006).
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