1: Nucleic Acids Res. 2005 Sep 16;33(16):5271-90. Print 2005. 

Binding of serum response factor to cystic fibrosis transmembrane conductance
regulator CArG-like elements, as a new potential CFTR transcriptional regulation
pathway.

Rene C, Taulan M, Iral F, Doudement J, L'Honore A, Gerbon C, Demaille J,
Claustres M, Romey MC.

Laboratoire de Genetique Moleculaire et Chromosomique, Institut Universitaire de
Recherche Clinique, Montpellier, France.

CFTR expression is tightly controlled by a complex network of ubiquitous and
tissue-specific cis-elements and trans-factors. To better understand mechanisms
that regulate transcription of CFTR, we examined transcription factors that
specifically bind a CFTR CArG-like motif we have previously shown to modulate
CFTR expression. Gel mobility shift assays and chromatin immunoprecipitation
analyses demonstrated the CFTR CArG-like motif binds serum response factor both
in vitro and in vivo. Transient co-transfections with various SRF expression
vector, including dominant-negative forms and small interfering RNA,
demonstrated that SRF significantly increases CFTR transcriptional activity in
bronchial epithelial cells. Mutagenesis studies suggested that in addition to
SRF other co-factors, such as Yin Yang 1 (YY1) previously shown to bind the CFTR
promoter, are potentially involved in the CFTR regulation. Here, we show that
functional interplay between SRF and YY1 might provide interesting perspectives
to further characterize the underlying molecular mechanism of the basal CFTR
transcriptional activity. Furthermore, the identification of multiple CArG
binding sites in highly conserved CFTR untranslated regions, which form specific
SRF complexes, provides direct evidence for a considerable role of SRF in the
CFTR transcriptional regulation into specialized epithelial lung cells.

PMID: 16170155 [PubMed - indexed for MEDLINE]


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2: Genes Dev. 2004 Nov 1;18(21):2627-38. 

Erratum in:
    Genes Dev. 2005 Mar 15;19(6):768.

The Polycomb Ezh2 methyltransferase regulates muscle gene expression and
skeletal muscle differentiation.

Caretti G, Di Padova M, Micales B, Lyons GE, Sartorelli V.

Muscle Gene Expression Group, Laboratory of Muscle Biology, NIAMS, National
Institutes of Health, Bethesda, Maryland 20892, USA.

The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine
methyltransferase (HKMT) activity that is associated with transcriptional
repression. We report that Ezh2 expression was developmentally regulated in the
myotome compartment of mouse somites and that its down-regulation coincided with
activation of muscle gene expression and differentiation of
satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle
differentiation, and this property was conferred by its SET domain, required for
the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated
with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with
the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their
presence correlated with methylation of K27 of histone H3. YY1 was required for
Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of
Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and
YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF
were 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.

PMID: 15520282 [PubMed - indexed for MEDLINE]


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3: Development. 2004 Feb;131(3):669-79. Epub 2004 Jan 7. 

Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus
embryonic development.

Latinkic BV, Cooper B, Smith S, Kotecha S, Towers N, Sparrow D, Mohun TJ.

Division of Developmental Biology, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, UK.

The mechanisms by which transcription factors, which are not themselves tissue
restricted, establish cardiomyocyte-specific patterns of transcription in vivo
are unknown. Nor do we understand how positional cues are integrated to provide
regionally distinct domains of gene expression within the developing heart. We
describe regulation of the Xenopus XMLC2 gene, which encodes a regulatory myosin
light chain of the contractile apparatus in cardiac muscle. This gene is
expressed from the onset of cardiac differentiation in the frog embryo and is
expressed throughout all the myocardium, both before and after heart chamber
formation. Using transgenesis in frog embryos, we have identified an 82 bp
enhancer within the proximal promoter region of the gene that is necessary and
sufficient for heart-specific expression of an XMLC2 transgene. This enhancer is
composed of two GATA sites and a composite YY1/CArG-like site. We show that the
low-affinity SRF site is essential for transgene expression and that
cardiac-specific expression also requires the presence of at least one adjacent
GATA site. The overlapping YY1 site within the enhancer appears to act primarily
as a repressor of ectopic expression, although it may also have a positive role.
Finally, we show that the frog MLC2 promoter drives pan myocardial expression of
a transgene in mice, despite the more restricted patterns of expression of
murine MLC2 genes. We speculate that a common regulatory mechanism may be
responsible for pan-myocardial expression of XMLC2 in both the frog and mouse,
modulation of which could have given rise to more restricted patterns of
expression within the heart of higher vertebrates.

PMID: 14711876 [PubMed - indexed for MEDLINE]


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4: Mol Biol Cell. 2003 May;14(5):2151-62. Epub 2003 Jan 26. 

MyoD distal regulatory region contains an SRF binding CArG element required for
MyoD expression in skeletal myoblasts and during muscle regeneration.

L'honore A, Lamb NJ, Vandromme M, Turowski P, Carnac G, Fernandez A.

Cell Biology Unit, Institut de Genetique Humaine, 34396 Montpellier cedex 05,
France.

We show here that the distal regulatory region (DRR) of the mouse and human MyoD
gene contains a conserved SRF binding CArG-like element. In electrophoretic
mobility shift assays with myoblast nuclear extracts, this CArG sequence,
although slightly divergent, bound two complexes containing, respectively, the
transcription factor YY1 and SRF associated with the acetyltransferase CBP and
members of C/EBP family. A single nucleotide mutation in the MyoD-CArG element
suppressed binding of both SRF and YY1 complexes and abolished DRR enhancer
activity in stably transfected myoblasts. This MyoD-CArG sequence is active in
modulating endogeneous MyoD gene expression because microinjection of
oligonucleotides corresponding to the MyoD-CArG sequence specifically and
rapidly suppressed MyoD expression in myoblasts. In vivo, the expression of a
transgenic construct comprising a minimal MyoD promoter fused to the DRR and
beta-galactosidase was induced with the same kinetics as MyoD during mouse
muscle regeneration. In contrast induction of this reporter was no longer seen
in regenerating muscle from transgenic mice carrying a mutated DRR-CArG. These
results show that an SRF binding CArG element present in MyoD gene DRR is
involved in the control of MyoD gene expression in skeletal myoblasts and in
mature muscle satellite cell activation during muscle regeneration.

PMID: 12802082 [PubMed - indexed for MEDLINE]


---------------------------------------------------------------


5: Biochem J. 2002 Jun 1;364(Pt 2):547-54. 

Increased actin polymerization reduces the inhibition of serum response factor
activity by Yin Yang 1.

Ellis PD, Martin KM, Rickman C, Metcalfe JC, Kemp PR.

Section of Cardiovascular Biology, Department of Biochemistry, University of
Cambridge, Building O, Downing Site, Cambridge CB2 1QW, UK.

Recent evidence has implicated CC(A/T(richG))GG (CArG) boxes, binding sites for
serum response factor (SRF), in the regulation of expression of a number of
genes in response to changes in the actin cytoskeleton. In many cases, the
activity of SRF at CArG boxes is modulated by transcription factors binding to
overlapping (e.g. Yin Yang 1, YY1) or adjacent (e.g. ets) binding sites.
However, the mechanisms by which SRF activity is regulated by the cytoskeleton
have not been determined. To investigate these mechanisms, we screened for cells
that did or did not increase the activity of a fragment of the promoter for a
smooth-muscle (SM)-specific gene SM22alpha, in response to changes in actin
cytoskeletal polymerization induced by LIM kinase. These experiments showed that
vascular SM cells (VSMCs) and C2C12 cells increased the activity of promoters
containing at least one of the SM22alpha CArG boxes (CArG near) in response to
LIM kinase, whereas P19 cells did not. Bandshift assays using a probe to CArG
near showed that P19 cells lacked detectable YY1 DNA binding to the CArG box in
contrast with the other two cell types. Expression of YY1 in P19 cells inhibited
SM22alpha promoter activity and conferred responsiveness to LIM kinase. Mutation
of the CArG box to inhibit YY1 or SRF binding indicated that both factors were
required for the LIM kinase response in VSMCs and C2C12 cells. The data indicate
that changes in the actin cytoskeletal organization modify SRF activity at CArG
boxes by modulating YY1-dependent inhibition.

PMID: 12023898 [PubMed - indexed for MEDLINE]


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6: J Biomed Sci. 1995 Aug;2(3):203-226. 

Multiple Tandemly Repeated Binding Sites for the YY1 Repressor and Transcription
Factors AP-1 and SP-1 Are Clustered within Intron-1 of the Gene Encoding the
IE110 Transactivator of Herpes simplex Virus Type 1.

Gu W, Huang Q, Hayward GS.

Department of Pharmacology and Molecular Sciences, Johns Hopkins University
School of Medicine, Baltimore, Md., USA.

Expression of the IE110 (ICP0) transactivator protein of HSV appears to be
critical for reactivation from the latent state and occurs at immediate-early
times during the lytic cycle under the control of an upstream divergent
enhancer-promoter region that contains multiple Oct and Sp-1 binding sites
overlapping with VP16 response elements. Surprisingly, the large 800-bp first
intron of the HSV-1 IE110 gene also proved to have a complex repetitive
organization encompassing multiple transcription factor binding sites within
four distinct domains. DNaseI footprinting studies revealed that 13 of 17 copies
of a 15-bp repeated element represented high-affinity binding sites for the
cellular YY1 repressor protein. Between 4 and 7 of these sites are direct tandem
repeats and the rest are interpersed with three repeated AT-rich motifs and a
dyad symmetry region containing two strong AP-1 binding sites and an adjacent
SP-1 binding site on each arm. Several of the YY1 sites also bound weakly to
SRF. The intron also contains four clustered purine/pyrimidine tracts of between
16 and 23 bp long. Both the AP-1/AP-2/SP-1 dyad protein binding region and, to a
lesser extent, the YY1 tandem-repeat cluster conferred responsiveness to TPA
when placed upstream of a heterologous promoter in transient expression assays.
The functional significance of the HSV-1 IE110 intron region is unknown as yet,
but the novel arrangement of tandemly repeated YY1 sites has the potential to
produce structural bending and transcriptional attenuation effects.
Interestingly, few of these transcription factor binding motifs are conserved in
the equivalent IE110 intron of HSV-2, and the domain appears to represent a
unique alternative control region that is specific for HSV-1. Copyright 1995 S.
Karger AG, Basel

PMID: 11725057 [PubMed - as supplied by publisher]


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7: J Biol Chem. 2001 May 11;276(19):16418-24. Epub 2001 Feb 8. 

Binding of serum response factor to CArG box sequences is necessary but not
sufficient to restrict gene expression to arterial smooth muscle cells.

Strobeck M, Kim S, Zhang JC, Clendenin C, Du KL, Parmacek MS.

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104, USA.

Serum response factor (SRF) plays an important role in regulating smooth muscle
cell (SMC) development and differentiation. To understand the molecular
mechanisms underlying the activity of SRF in SMCs, the two CArG box-containing
elements in the arterial SMC-specific SM22alpha promoter, SME-1 and SME-4, were
functionally and biochemically characterized. Mutations that abolish binding of
SRF to the SM22alpha promoter totally abolish promoter activity in transgenic
mice. Moreover, a multimerized copy of either SME-1 or SME-4 subcloned 5' of the
minimal SM22alpha promoter (base pairs -90 to +41) is necessary and sufficient
to restrict transgene expression to arterial SMCs in transgenic mice. In
contrast, a multimerized copy of the c-fos SRE is totally inactive in arterial
SMCs and substitution of the c-fos SRE for the CArG motifs within the SM22alpha
promoter inactivates the 441-base pair SM22alpha promoter in transgenic mice.
Deletion analysis revealed that the SME-4 CArG box alone is insufficient to
activate transcription in SMCs and additional 5'-flanking nucleotides are
required. Nuclear protein binding assays revealed that SME-4 binds SRF, YY1, and
four additional SMC nuclear proteins. Taken together, these data demonstrate
that binding of SRF to specific CArG boxes is necessary, but not sufficient, to
restrict transgene expression to SMCs in vivo.

PMID: 11279108 [PubMed - indexed for MEDLINE]


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8: J Mol Cell Cardiol. 2001 Jan;33(1):95-107. 

Nitric oxide regulates smooth-muscle-specific myosin heavy chain gene expression
at the transcriptional level-possible role of SRF and YY1 through CArG element.

Itoh S, Katoh Y, Konishi H, Takaya N, Kimura T, Periasamy M, Yamaguchi H.

Department of Cardiology, Juntendo University School of Medicine, 2-1-1 Hongo,
Bunkyo-ku, Tokyo, 113-8421, Japan.

Nitric oxide (NO) plays an important role in vascular regulation through its
vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits
platelet adhesion and aggregation and modulates smooth muscle cell (SMC)
proliferation and migration. In animals with experimentally induced vascular
injury, ec-NOS gene transfection not only restored NO production to normal
levels but also increased vascular reactivity of the injured vessels. However,
it is unclear whether NO regulates smooth-muscle-specific gene expression. We
report here that addition of PDGF-BB to vascular smooth muscle cells suppressed
SM-MHC expression but treatment with the NO donors FK409 and SNAP restored
SM-MHC mRNA/protein expression. In vitro transfection and subsequent CAT assays
demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC
promoter activity. Promoter deletion analysis revealed that a CArG-3 box located
at -1276 bp in the SM-MHC promoter was important for NO-dependent promoter
regulation and as well as high level promoter activity. Gel mobility shift
assays showed that CArG-3 contained the SRF binding site and a binding site for
YY1, a nuclear factor which acts as a negative regulator on muscle-specific
promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3
element but increased SRF binding, suggesting that these two factors bind
competitively to the overlapping sites. We also found that mutation to the YY1
binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced
suppression of the SM-MHC promoter activity. These findings indicate that NO
regulates SM-MHC gene expression at the transcriptional level at least partially
through the regulation of transcription factor binding activities on the CArG
element. Thus we propose that NO plays a positive role in maintaining the
differentiated state of VSMCs.

PMID: 11133226 [PubMed - indexed for MEDLINE]


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9: Mamm Genome. 2000 Dec;11(12):1053-7. 

Genomic organization and mapping of mouse CDV (carnitine deficiency-associated
gene expressed in ventricle)-1 and its related CDV-1R gene.

Higashi M, Kobayashi K, Iijima M, Wakana S, Horiuchi M, Yasuda T, Yoshida G,
Kanmura Y, Saheki T.

Department of Biochemistry, Faculty of Medicine, Kagoshima University,
Sakuragaoka, Japan.

We have previously reported that CDV (carnitine deficiency-associated gene
expressed in ventricle)-1 was a downregulated gene in the hypertrophied
ventricle of carnitine-deficient juvenile visceral steatosis mice and that the
related gene (CDV-1R) showed no tissue specificity and no sensitivity to
carnitine deficiency. In the present paper, the CDV-1/1R gene was isolated from
a mouse genomic BAC library, and the genomic structure was characterized. We
found that the CDV-1/1R gene consisted of at least 19 exons and encompassed
approximately 48 kb. The splice sites conformed to the GT-AG rule, and the
CDV-1R mRNA containing 19 exons was processed. CDV-1 mRNA containing 5 exons was
constructed from the 3' half of CDV-1R. The first exon of CDV-1 consisted of the
3' side (116 bp) of intron 14 and exon 15 (87 bp) of CDV-1R. The presumed
promoter sequence for CDV-1 located in the intron 14 of CDV-1R contained the
common TATA box and consensus binding sites for various transcription factors
(Nkx-2.5, Spl, C/EBP, SRF, YY1, and CREB), which seem to play roles in the
heart-specific expression and carnitine deficiency-associated suppression of
CDV-1. In the upstream region of the CDV-1 promoter, we found two VNTRs, 13
repeats of GATA1, and 16 copies of STRE involved in yeast stress response. The
CDV-1/1R gene was located close to DSMIT68 on mouse Chromosome (Chr) 5,
corresponding to human Chr 12q24. All these data revealed that two mRNA species,
CDV-1 and CDV-1R, are expressed tissue-specifically by using promoters peculiar
to each transcript in a single gene.

PMID: 11130971 [PubMed - indexed for MEDLINE]


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10: Gene Expr. 1999;8(1):33-42. 

Autonomously binding protein detected on ets box of c-fos serum response element
in proliferating cells.

Masutani H, Magnaghi-Jaulin L, Groisman R, Ait-Si-Ali S, Robin P, Pritchard LL,
Harel-Bellan A.

Laboratoire de Biologie des Tumeurs Humaines, CNRS URA 1156, Institut Gustave
Roussy, Villejuif, France.

The serum response element (SRE) in the c-fos promoter contains an ets box whose
integrity is required for full activation of this proto-oncogene by nerve growth
factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift
assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type
SRE, but not to an SRE containing a mutated ets box. Competition studies using
unlabeled probes, and supershift experiments using antibodies and in vitro
translated core serum response factor (SRF) indicate that the protein in
question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary
complex factor (TCF) activity, so that it may correspond to an autonomously
binding Ets family protein. The complete disappearance of this "Ets-like
autonomous binding factor" upon terminal differentiation of both L6alpha2
myoblastic and PC12 pheochromocytoma cells points to a possible role in the
proliferation/differentiation process.

PMID: 10543729 [PubMed - indexed for MEDLINE]


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11: Nucleic Acids Res. 1998 Jul 1;26(13):3215-20. 

Influence of promoter potency on the transcriptional effects of YY1, SRF and
Msx-1 in transient transfection analysis.

Lee T, Bradley ME, Walowitz JL.

Department of Biochemistry, SUNY at Buffalo, 140 Farber Hall, 3435 Main Street,
Buffalo, NY 14214-3000, USA. chunglee@acsu.buffalo.edu

Potent viral promoters/enhancers are often used to achieve high level expression
of ectopic genes in transient transfection analysis. By using a GAL4-responsive
transcription assay system, we show that the use of potent eukaryotic expression
vectors can lead to biased transcriptional effects. Three functionally diverse
transcription factors, YY1, SRF and Msx-1, were examined and each was found to
exhibit a strong transrepression function in the context of the DNA binding
domain of GAL4 when expressed from the cytomegalovirus (pCMV) or simian virus 40
(pSV) promoters/enhancers. An internal 15 amino acid domain of YY1 mediating
transrepression in the viral promoter setting was identified. This GAL4-mediated
transcriptional repression could, however, be completely relieved by using the
yeast alcohol dehydrogenase promoter (pADH) to drive gene expression, which is
approximately 100-fold weaker than canonical pCMV and pSV in cultured mammalian
cells. In addition, low level expression achieved with the pADH vector unveiled
the intrinsic transactivation functions of YY1 and SRF previously not observed
with the GAL4 assay system. Our results highlight a potential pitfall in
conventional pCMV- and pSV-based transfection assays and suggest that the use of
a low level expression system may be preferable in most transcriptional
analysis.

PMID: 9628921 [PubMed - indexed for MEDLINE]


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12: Mol Endocrinol. 1997 Jun;11(6):812-22. 

Competition between negative acting YY1 versus positive acting serum response
factor and tinman homologue Nkx-2.5 regulates cardiac alpha-actin promoter
activity.

Chen CY, Schwartz RJ.

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030,
USA.

Transcription of sarcomeric alpha-actin genes is developmentally regulated
during skeletal and cardiac muscle development through fine-tuned control
mechanisms involving multiple cooperative and antagonistic transcription
factors. Among the cis-acting DNA elements recognized by these factors is the
sequence CC(A/T)6GG of the serum response element (SRE), which is present in a
number of growth factor-inducible and myogenic specified genes. We recently
showed that the cardiogenic homeodomain factor, Nkx-2.5, served as a positive
acting accessory factor for serum response factor (SRF) and together provided
strong transcriptional activation of the cardiac alpha-actin promoter. In
addition, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac
alpha-actin gene in 10T1/2 fibroblasts, by a mechanism that involved
coassociation of SRF and Nkx-2.5 on intact SREs of the alpha-actin promoter.
Here, we show that the second SRE of the avian cardiac alpha-actin promoter
served as a binding site for Nkx-2.5, SRF, and zinc finger containing
GLI-Kruppel-like factor, YY1. Expression of YY1 inhibited cardiac alpha-actin
promoter activity, whereas coexpression of Nkx-2.5 and SRF was able to partially
reverse YY1 repression. Displacement of YY1 binding by Nkx-2.5/SRF complex
occurs through mutually exclusive binding across the CaSRE2. The interplay and
functional antagonism between YY1 and Nkx-2.5/SRF might constitute a
developmental as well as a physiologically regulated mechanism that modulates
cardiac alpha-actin gene expression during cardiogenesis.

PMID: 9171244 [PubMed - indexed for MEDLINE]


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13: DNA Cell Biol. 1997 May;16(5):653-61. 

A competitive mechanism of CArG element regulation by YY1 and SRF: implications
for assessment of Phox1/MHox transcription factor interactions at CArG elements.

Martin KA, Gualberto A, Kolman MF, Lowry J, Walsh K.

Department of Physiology and Biophysics, School of Medicine, Case Western
Reserve University, Cleveland, OH 44106, USA.

In the promoters of many immediate early genes, including c-fos, CArG DNA
regulatory elements mediate basal constituitive expression and rapid and
transient serum induction. CArG boxes also occur in the promoters of
muscle-specific genes, including skeletal alpha-actin, where it confers
muscle-specific expression. These elements are regulated, at least in part, by
the ubiquitous transcription factors serum response factor (SRF) and YY1. The
homeobox transcription factor Phox1/MHox has also been implicated in regulation
of the c-fos CArG element and is thought to function by facilitating SRF binding
to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1
repression of CArG elements results from competition with SRF for overlapping
binding sites. We describe in detail the binding sites of YY1 and SRF through
serial point mutations of the skeletal alpha-actin proximal CArG element and
identify a mutation that dramatically reduces YY1 binding but retains normal SRF
binding. YY1 competes with SRF for binding to wild-type CArG elements, but not
to this point mutant in vitro. This mutant is sufficient for muscle-specific
expression in vivo but is much less sensitive to repression by YY1
overexpression. We utilized the YY1/SRF competition to address the role of Phox1
at these elements. Phox1 overexpression did not diminish YY1-mediated
repression, suggesting that transcriptional activation by Phox1 does not result
from enhanced SRF binding to these elements. These methods may prove to be
useful for assessing interactions between other CArG element regulatory factors.

PMID: 9174170 [PubMed - indexed for MEDLINE]


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14: J Virol. 1996 Dec;70(12):8590-605. 

Synergistic interactions between overlapping binding sites for the serum
response factor and ELK-1 proteins mediate both basal enhancement and phorbol
ester responsiveness of primate cytomegalovirus major immediate-early promoters
in monocyte and T-lymphocyte cell types.

Chan YJ, Chiou CJ, Huang Q, Hayward GS.

Department of Pharmacology, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205, USA.

Cytomegalovirus (CMV) infection is nonpermissive or persistent in many lymphoid
and myeloid cell types but can be activated in differentiated macrophages. We
have shown elsewhere that both the major immediate-early gene (MIE) and lytic
cycle infectious progeny virus expression can be induced in otherwise
nonpermissive monocyte-like U-937 cell cultures infected with either human CMV
(HCMV) or simian CMV (SCMV) by treatment with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA). Two multicopy basal enhancer motifs
within the SCMV MIE enhancer, namely, 11 copies of the 16-bp cyclic AMP response
element (CRE) and 3 copies of novel 17-bp serum response factor (SRF) binding
sites referred to as the SNE (SRF/NFkappaB-like element), as well as four
classical NFkappaB sites within the HCMV version, contribute to TPA
responsiveness in transient assays in monocyte and T-cell types. The SCMV SNE
sites contain potential overlapping core recognition binding motifs for SRF,
Rel/NFkappaB, ETS, and YY1 class transcription factors but fail to respond to
either serum or tumor necrosis factor alpha. Therefore, to evaluate the
mechanism of TPA responsiveness of the SNE motifs and of a related 16-bp SEE
(SRF/ETS element) motif found in the HCMV and chimpanzee CMV MIE enhancers, we
have examined the functional responses and protein binding properties of
multimerized wild-type and mutant elements added upstream to the SCMV MIE or
simian virus 40 minimal promoter regions in the U-937, K-562, HL-60, THP-1, and
Jurkat cell lines. Unlike classical NFkappaB sites, neither the SNE nor the SEE
motif responded to phosphatase inhibition by okadaic acid. However, the TPA
responsiveness of both CMV elements proved to involve synergistic interactions
between the core SRF binding site (CCATATATGG) and the adjacent inverted ETS
binding motifs (TTCC), which correlated directly with formation of a bound
tripartite complex containing both the cellular SRF and ELK-1 proteins. This
protein complex was more abundant in U-937, K-562, and HeLa cell extracts than
in Raji, HF, BALB/c 3T3, or HL-60 cells, but the binding activity was altered
only twofold after TPA treatment. A 40-fold stimulation of chloramphenicol
acetyltransferase activity mediated by four tandem repeats of the SNE could be
induced within 2 h (and up to 250-fold within 6 h) after addition of TPA in
DNA-transfected U-937 cells, indicating that the stimulation appeared likely to
be a true protein kinase C-mediated signal transduction event rather than a
differentiation response. Slight differences in the sequence of the core SRF
binding site compared with that of the classical c-Fos promoter serum response
element, together with differences in the spacing between the SRF and ETS
motifs, appear to account for the inability of the SCMV SNEs to respond to serum
induction.

PMID: 8970984 [PubMed - indexed for MEDLINE]


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15: J Biol Chem. 1996 May 3;271(18):10827-33. 

Serum response factor mediates AP-1-dependent induction of the skeletal
alpha-actin promoter in ventricular myocytes.

Paradis P, MacLellan WR, Belaguli NS, Schwartz RJ, Schneider MD.

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA.

"Fetal" gene transcription, including activation of the skeletal alpha-actin
(SkA) promoter, is provoked in cardiac myocytes by mechanical stress and trophic
ligands. Induction of the promoter by transforming growth factor beta or
norepinephrine requires serum response factor (SRF) and TEF-1; expression is
inhibited by YY1. We and others postulated that immediate-early transcription
factors might couple trophic signals to this fetal program. However, multiple
Fos/Jun proteins exist, and the exact relationship between control by Fos/Jun
versus SRF, TEF-1, and YY1 is unexplained. We therefore cotransfected
ventricular myocytes with Fos, Jun, or JunB, and SkA reporter genes. SkA
transcription was augmented by Jun, Fos/Jun, Fos/JunB, and Jun/JunB; Fos and
JunB alone were neutral or inhibitory. Mutation of the SRF site, SRE1, impaired
activation by Jun; YY1, TEF-1, and Sp1 sites were dispensable. SRE1 conferred
Jun activation to a heterologous promoter, as did the c-fos SRE. Deletions of
DNA binding, dimerization, or trans-activation domains of Jun and SRF abolished
activation by Jun and synergy with SRF. Neither direct binding of Fos/Jun to
SREs, nor physical interaction between Fos/Jun and SRF, was detected in
mobility-shift assays. Thus, AP-1 factors activate a hypertrophy-associated gene
via SRF, without detectable binding to the promoter or to SRF.

PMID: 8631897 [PubMed - indexed for MEDLINE]


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16: Mol Cell Biol. 1995 Nov;15(11):5975-82. 

YY1 facilitates the association of serum response factor with the c-fos serum
response element.

Natesan S, Gilman M.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

YY1 is a multifunctional transcription factor that acts as an activator or
repressor in different contexts. YY1 binds to multiple sites in the mouse c-fos
promoter, inducing at each site a sharp DNA bend. Binding of YY1 to a site
situated between the cyclic AMP response element (CRE) and the TATA box bends
the DNA in a way that interferes with the interaction of proteins bound at the
CRE and TATA elements, resulting in repression of transcription. Here, we show
that binding of YY1 to a different site in the c-fos promoter has a different
result. Binding of YY1 to the c-fos serum response element (SRE) enhances the
binding of serum response factor (SRF). This enhancement requires the binding of
YY1 to SRE DNA. YY1 and SRF can cooccupy the SRE at least transiently. In the
region of overlapping contact, YY1 contacts DNA in the major groove, while SRF
contacts DNA in the minor groove. YY1 also enhances the association of SRF with
the SRE in transfected insect cells. Thus, although YY1 induces similar
structural changes in DNA at different binding sites, it can have distinct local
effects on protein-DNA and protein-protein interactions. These data support a
general role for YY1 in the building of highly organized promoter complexes.

PMID: 7565750 [PubMed - indexed for MEDLINE]


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17: Oncogene. 1995 Apr 6;10(7):1361-70. 

Two serum response elements mediate transcriptional repression of human smooth
muscle alpha-actin promoter in ras-transformed cells.

Bushel P, Kim JH, Chang W, Catino JJ, Ruley HE, Kumar CC.

Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, New
Jersey 07033, USA.

The mechanism by which activated ras oncogene expression leads to repression of
genes encoding specific actin filament proteins is not understood. However,
these changes associated with loss of organized actin filaments, are necessary
to maintain the transformed phenotype. The human smooth muscle (sm) alpha-actin
promoter is repressed in ras-transformed fibroblast cells and derepressed in
revertant cell lines. In this study, we demonstrate that two serum response
elements (SREs) present in the alpha-actin promoter are required for
transcriptional repression in ras-transformed cells and the two SREs act
synergistically to repress heterologous promoters in a ras-transformation
dependent manner. Serum response factor (SRF), which can bind to the sm
alpha-actin SREs, restores alpha-actin promoter activity in ras-transformed
cells. c-Fos, c-Jun and YY1 also repress alpha-actin promoter through SREs,
suggesting that these transcription factors may play a role in repressing
alpha-actin promoter in ras-transformed cells.

PMID: 7731687 [PubMed - indexed for MEDLINE]


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18: Dev Genet. 1995;16(3):229-40. 

Serum response element associated transcription factors in mouse embryos: serum
response factor, YY1, and PEA3 factor.

Liu SH, Peng BH, Ma JT, Liu YC, Ng SY.

Institute of Molecular Biology, Academia Sinica, NanKang, Taipei, Republic of
China.

Many mammalian transcription factors, including human and mouse serum response
factors (SRFs), are post-translationally modified with O-linked
N-acetylglucosamine monosaccharides on multiple serine and/or threonine
residues. Nuclear extracts were prepared from 9.5 to 19 days postcoitum mouse
embryos and subsequently were fractionated by wheat germ agglutinin
(WGA)-agarose affinity chromatography. SRF binds WGA-agarose and apparently is
O-glycosylated. On the other hand, the low molecular weight serum response
element (SRE)-binding proteins, including the previously named band I and band
II factors, did not bind WGA-agarose. Furthermore, we showed that the fastest
migrating complex contains the Yin-Yang 1 (YY1) factor. YY1 binds to the c-fos
SRE and skeletal alpha-actin muscle regulatory element (MRE), but not the
cardiac alpha-actin MRE. Nuclear extracts from NIH/3T3 fibroblasts contain
similar, if not identical, SRE-binding complexes. Besides these SRE-binding
factors, mouse PEA3-binding factor, presumably an ETS domain-containing protein,
was found to bind SRF protein. This physical interaction, between SRF and ETS
domain proteins, was shown to involve the DNA-binding domain-containing region
of SRF and not the carboxyl-terminal transactivation domain.

PMID: 7796532 [PubMed - indexed for MEDLINE]


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19: Mol Reprod Dev. 1994 Sep;39(1):112-7. 

Control of cardiac gene transcription by fibroblast growth factors.

Schneider MD, Kirshenbaum LA, Brand T, MacLellan WR.

Molecular Cardiology Unit, Baylor College of Medicine, Houston, Texas 77030.

Skeletal alpha-actin (SkA) is representative of the cardiac genes that are
expressed at high levels in embryonic myocardium, downregulated after birth, and
reactivated by tropic signals including basic fibroblast growth factor (FGF-2)
and type beta transforming growth factors (TGF beta). To investigate the
molecular basis for cardiac-restricted and growth factor-induced SkA
transcription, we have undertaken a mutational analysis of the SkA promoter in
neonatal ventricular myocytes, with emphasis on the role of three nominal serum
response elements. Serum response factor (SRF) and the bifunctional factor YY1
are the predominant cardiac proteins contacting the proximal SRE (SRE1).
Mutations of SRE1 that prevent recognition by SRF and YY1. or SRF alone,
virtually abolish SkA transcription; mutation of distal SREs was ineffective. A
mutation which selectively abrogates YY1 binding increases expression,
substantiating the predicted role of YY1 as an inhibitor of SRF effects. SkA
transcription requires combinational action of SRE1 with consensus sites for Sp1
and the SV40 enhancer binding protein, TEF-1. As an isolated motif, SRE1 can
confer responsiveness to both FGF-2 and TGF beta to a heterologous promoter.
Whether TEF-1 binding sites likewise can function as FGF response elements is
unknown. Molecular dissection of mechanisms that govern the differentiated
cardiac phenotype has largely been undertaken to date in neonatal ventricular
myocytes, as the adult ventricular myocyte has been refractory to conventional
procedures for gene transfer. To circumvent expected limitations of other
methods, we have used replication-deficient adenovirus to achieve efficient gene
transfer to adult cardiac cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication Types:
    Review
    Review, Tutorial

PMID: 7528025 [PubMed - indexed for MEDLINE]


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20: J Biol Chem. 1994 Jun 17;269(24):16754-60. 

Transforming growth factor-beta response elements of the skeletal alpha-actin
gene. Combinatorial action of serum response factor, YY1, and the SV40
enhancer-binding protein, TEF-1.

MacLellan WR, Lee TC, Schwartz RJ, Schneider MD.

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.

Skeletal alpha-actin (SkA) is representative of the cardiac genes that are
expressed at high levels in embryonic myocardium, down-regulated after birth,
and reactivated by trophic signals including type beta-transforming growth
factors (TGF beta). To investigate the molecular basis for cardiac-restricted
and TGF beta-induced SkA transcription, we have undertaken a mutational analysis
of the SkA promoter in ventricular myocytes, with emphasis on the role of three
nominal serum response elements. Serum response factor (SRF) and the
bifunctional factor YY1 are the predominant cardiac proteins contacting the
proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1,
or SRF alone, virtually abolish SkA transcription in both TGF beta- and
vehicle-treated cells; mutation of distal SREs was ineffective. A mutation which
selectively abrogates YY1 binding increases both basal and TGF beta-dependent
expression, substantiating the predicted role of YY1 as an inhibitor of SRF
effects. However, efficient SkA transcription requires combinatorial action of
SRE1 with consensus sites for Sp1 and the SV40 enhancer-binding protein, TEF-1.
As isolated motifs, either SRE1- or TEF-1-binding sites function as TGF beta
response elements. Induction of the SkA promoter by TGF beta required SRF and
TEF-1 in concert, unlike other pathways for TGF beta-dependent gene expression.

PMID: 8206998 [PubMed - indexed for MEDLINE]


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21: Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9814-8. 

Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal
alpha-actin transcription in embryonic myoblasts.

Lee TC, Shi Y, Schwartz RJ.

Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

Muscle-restricted transcription of the skeletal alpha-actin gene is controlled
in part by a positive regulator, serum response factor (SRF), and a negative
regulator, F-ACT1, which bind competitively to the most proximal serum response
element (SRE1). We show here that F-ACT1 is identical to a transcription factor
recently cloned and described as YY1, NF-E1, delta, or UCRBP. We found that
although the DNA-binding activity of SRF accumulates during myogenesis, that of
YY1 diminishes simultaneously. Myoblasts rendered incapable of differentiation
by BrdUrd treatment exhibited the highest level of YY1 and the lowest level of
SRF activities. Transfected SRF could directly transactivate the skeletal
alpha-actin promoter by overcoming the inhibitory effect of BrdUrd-induced YY1.
The transactivation depends on intact SRE DNA elements and requires the
DNA-binding/dimerization domain of SRF as well as its C-terminal half rich in
serines and threonines. Since the functions of YY1 and SRF appear to be
developmentally regulated, the convergence of their binding sites upon the SRE
constitutes an integrated mechanism whereby temporal and spatial muscle gene
expression may be accomplished.

PMID: 1409704 [PubMed - indexed for MEDLINE]


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22: Mol Cell Biol. 1992 Sep;12(9):4209-14. 

Functional antagonism between YY1 and the serum response factor.

Gualberto A, LePage D, Pons G, Mader SL, Park K, Atchison ML, Walsh K.

Department of Physiology and Biophysics, Case Western Reserve University School
of Medicine, Cleveland, Ohio 44106.

The rapid, transient induction of the c-fos proto-oncogene by serum growth
factors is mediated by the serum response element (SRE). The SRE shares homology
with the muscle regulatory element (MRE) of the skeletal alpha-actin promoter.
It is not known how these elements respond to proliferative and
cell-type-specific signals, but the response appears to involve the binding of
the serum response factor (SRF) and other proteins. Here, we report that YY1, a
multifunctional transcription factor, binds to SRE and MRE sequences in vitro.
The methylation interference footprint of YY1 overlaps with that of the SRF, and
YY1 competes with the SRF for binding to these DNA elements. Overexpression of
YY1 repressed serum-inducible and basal expression from the c-fos promoter and
repressed basal expression from the skeletal alpha-actin promoter. YY1 also
repressed expression from the individual SRE and MRE sequences upstream from a
TATA element. Unlike that of YY1, SRF overexpression alone did not influence the
transcriptional activity of the target sequence, but SRF overexpression could
reverse YY1-mediated trans repression. These data suggest that YY1 and the SRF
have antagonistic functions in vivo.

PMID: 1508214 [PubMed - indexed for MEDLINE]


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