BACKGROUND
the protein p49/strap was identified in our laboratory in search of proteins that regulate cytoskeletal genes related to cardiac function  <cit> .
our initial observations indicated that p49/strap is a cofactor of serum response factor , contributing to the regulation of srf target genes.
this gene was later named by the human genome organization  as srfbp <dig>  based on our initial finding that p49/strap binds to srf.
serum response factor is an important transcription factor that regulates a number of immediate-early genes, cytoskeletal and muscle-specific genes.
srf has been reported to exhibit functional interactions with a number of srf cofactors and/or binding proteins in the regulation of srf target genes  <cit> .
these interactions likely modulate srf function and may also enable srf to mediate tissue-specific regulation of its target genes at different developmental stages  <cit> .
a model of transcriptional regulation by srf and its binding proteins has been proposed, in which the interaction of multiple proteins  affects the outcome of the expression of srf target genes  <cit> .
srf has two functional domains.
the n-terminus is a dna binding domain that binds to the serum response element or carg box and the c-terminus is a transactivation domain  <cit> .
the p49/strap protein interacts directly with the srf transcriptional activation domain.
therefore, the level of p49/strap expression and its affinity to srf protein may have a significant impact on srf activation  <cit> .
the p49/strap mrna and protein are expressed in multiple tissues, including heart, brain, liver, skeletal muscle and kidney  <cit> .
an increase of p49/strap expression has been observed in the myocardium of adult human and mouse with advancing age, and also in transgenic mouse models of cardiomyopathy  <cit> .
however, the functional domains of the p49/strap protein and the effect of increased in-vitro and in-vivo p49/strap expression remain to be defined.
in the present study, the p49/strap protein sequence was analyzed for conserved domains.
a srf-binding domain was found at the n-terminus of the p49/strap proteins of mammals and vertebrates; and a bud <dig> domain was found at the c-terminus of the p49/strap proteins and the yeast cellular morphogenesis protein .
when p49/strap gene was overexpressed in cultured cells and in transgenic mice, morphological alterations were observed, both at the cellular and whole organ, structural level.
RESULTS
bioinformatics analysis of p49/strap protein
the p49/strap protein sequences of human, mouse and rat were analyzed using bioinformatics tools.
sequence alignment revealed that there was approximately 60% homology among human, mouse and rat p49/strap proteins.
further sequence alignment of  <dig> mammalian p49/strap proteins, including that of chimpanzee and dog, showed a 52% homology in p49/strap proteins.
two conserved regions were found at the n-terminus  and c-terminus  .
the conserved “n-region” overlaps with the “srf-binding domain” that was found to interact with srf in our previous study  <cit> .
the p49/strap protein sequences were compared against the “conserved domain database” at the national center for biotechnology information , the “pfam” and the interpro databases.
the c-region of p49/strap matched an existing conserved domain in all three databases, specifically the bud <dig> domain of the cellular morphogenesis protein that regulates yeast budding selection, polarity and rna biogenesis   <cit> .
each database showed a slightly different length of bud <dig> domain in p49/strap protein.
as for the human p49/strap , the bud <dig> domain was at amino acid 399– <dig> in the ncbi conserved domain database; amino acid 329– <dig> in the pfam database, and amino acid 360– <dig> in the interpro database, respectively.
alignment of the bud <dig> sequence of  <dig> yeast strands revealed that there was a conserved domain at the c-terminus , ranging from amino acid  <dig> to the end of the sequences .
when the p49/strap protein sequences of  <dig> mammalian species were compared with the  <dig> yeast bud <dig> proteins, it was found that only part of sequences at the end of c-region, approximately  <dig> amino acids, was homologous among the  <dig> sequences , indicating that those sequences represented a highly conserved region in the bud <dig> domain .
bioinformatics analysis indicated that there are four nls sequences in the human, mouse and rat p49/strap proteins, while three nls sequences were found within bud <dig> of s. cerevisiae and four nlss in bud <dig> of s. pombe.
it was also predicted that mouse p49/strap had two nucleolar localization sequences , and baker’s yeast bud <dig> had three nucleolar localization sequences .
in addition, it was also predicted that both p49/strap and bud <dig> proteins would be present in the cytoplasmic compartments, including mitochondria, endoplasmic reticulum, plasma membrane and vesicles of the secretory system .
both p <dig> and bud <dig> proteins were predicted to be present in the nucleus, but also in the cytoplasmic compartments, including mitochondria, endoplasmic reticulum, plasma membrane and vesicles of the secretory system.
intracellular distribution of p49/strap
we previously observed that transfected gfp-p49/strap fusion protein was mainly localized in the nucleus of nih3t <dig> cells, where it acts as a srf cofactor to regulate srf-target genes  <cit> .
indeed, bioinformatics analysis revealed that the mouse p49/strap had both nuclear localization signal sequences and nucleolar localization signal sequences .to confirm the bioinformatics result, h9c <dig> cells were transfected with the gfp-expression control plasmid and gfp-p49/strap expression plasmid, respectively.
as shown in figure 2a and c, the green gfp fluorescence was present in the entire cell in gfp-control plasmid transfected cell.
by contrast, in the gfp-p49/strap expression plasmid transfected cells, the gfp-p49/strap protein appeared to be present mainly in nucleus, with a few very bright dots within the nucleus .
we used nucleolin  to co-stain the transfected cells.
as shown in figure 2e, nucleolin was stained in red.
when figure 2d and e were merged , the bright green-colored gfp-p49/strap dots co-localized with the red-colored nucleolin , indicating that the p49/strap protein does localize within the nucleolus.
the p49/strap protein is known to be highly expressed in multiple tissues based on western blotting  <cit> .
to better understand the intracellular distribution of the endogenous p49/strap protein, an affinity-purified antibody against a p49/strap epitope was used for immunochemistry staining of h9c <dig> cells.
as shown in figure 2g, the endogenous p49/strap protein was present in both the nucleus and cytoplasm.
in addition, the p49/strap protein was widely distributed within the cell in close proximity to the actin fibers ; some of the p49/strap proteins appeared to form a “tail” that was co-located at the end of the actin fibers, close to their attachment sites to the inner wall of the cell membrane .
these findings suggest that p49/strap may play a role in cellular cytoskeletal structure and function via its presence in the cytoplasm.
p49/strap overexpression affects cell morphology
to determine the effect of p49/strap overexpression on cellular morphology, we infected h9c <dig> cells with the gfp-control adenovirus or recombinant p49/strap adenovirus  <cit> .
at  <dig> hours post-infection, the cells were fixed and immunocytochemistry staining was performed.
the phalloidin was used to stain f-actin.
the p49/strap antibody and alexa fluor  <dig> labelled secondary antibody were used to stain the cells to visualize the fluorescent intensity of both the endogenous and the exogenous p49/strap proteins in the p49/strap-virus treated cells versus that of the endogenous p49/strap protein only in the control cells infected with gfp-control virus.as shown in figure 3a-d, in the gfp-control virus infected cells, the actin fibers were present as thick, well aligned bundles , and the p <dig> protein was mainly localized in the nucleus ; however, in the p49/strap virus treated cells, the structure of the actin fibers was more diffuse, especially in the perinuclear region, and the intensity of the actin fibers was weaker .
asymmetric distribution of the p49/strap protein was observed within the p49/strap treated cells compared to that in the control cells .
cell size measurements revealed that h9c <dig> cells treated with the p49/strap adenovirus were smaller  compared to that of the control adenovirus infected cells .
in addition, p49/strap treated cells showed much larger nucleocytoplasmic ratios compared to that of the control, 1: <dig>  ±  <dig>  versus 1: <dig>  ±  <dig> , respectively .
the overall microscopic observation revealed that increased p49/strap protein reduced the f-actin structure and intensity, and changed the cell morphology in term of cytoskeletal structure, cell size, cell shape and nucleocytoplasmic ratio.
p49/strap modulated srf gene expression
p49/strap is a srf cofactor that co-regulates srf-target genes  <cit> .
since there are two srf-binding sites  in the srf gene promoter, the srf gene itself is also a srf-target gene subject to the regulation by srf  <cit> .
to test the hypothesis that p49/strap may regulate the srf gene, a plasmid containing firefly luciferase driven by the srf gene promoter was used in the luciferase-based promoter activity assay.
as shown in figure 5a, the srf plasmid transfection induced srf gene promoter activity ; when p49/strap was co-transfected with srf, p49/strap repressed the srf-induced promoter activity .
another srf cofactor, myocardin was also used in the transfection assay; myocardin overexpression induced srf gene promoter activity by approximately 5-fold , while p49/strap repressed the srf-promoter activity that was induced by myocardin plasmid transfection .
the mrna levels of srf and atrial natriuretic peptide  genes were also measured in response to p49/strap overexpression.
as shown in figure 5b, p49/strap repressed the expression of both the srf gene  and the anf gene .
generation of p49/strap transgenic mouse lines
the p49/strap mrna/protein is well expressed in multiple tissues, including brain, heart, skeletal muscle, kidney, liver and testis  <cit> .
to test the hypothesis that increased expression of p49/strap in vivo may alter the expression of srf-target genes and affect the morphology of mouse cells and tissues, we generated transgenic mouse lines containing a dna construct with p49/strap cdna under the transcriptional control of the cmv promoter.
after multiple microinjections and screenings, three transgenic  founder mice were identified.
the founder mice were bred to non-transgenic  mice, and three p49tg lines were thus established.
both southern blotting and quantitative pcr analysis were used for the detection of the transgene copy number of the three p49tg lines.
each line was found to have only one transgene copy.
death of some p49/strap transgenic newborn mice
the p49tg founder mice that survived to adulthood were fertile without apparent abnormality in reproduction.
the p49tg offspring had their genotype confirmed by pcr.
an examination of  <dig> litters from  <dig> generations of the p49tg mice revealed that the transgenic offspring separated into two groups based on p49/strap mrna expression level and their phenotype .
a close check of three mouse generations revealed that among  <dig> newborn p49tg mice,  <dig> newborns were malformed, which accounted for 43% of the p49tg offspring.
the malformed group of newborn p49tg mice  displayed serious defects and almost all of them were either dead at birth or died immediately thereafter .
these malformed mice had elevated level of p49/strap mrna expression .
the other group of newborn p49tg mice were grossly normal-appearing , had no apparent anatomic defects and most of these p49tg mice survived without visible abnormality.
these na-p49tg mice had similar levels of p49/strap mrna expression compared with that of the non-tg mice .
however, their p49/strap transgenic status was confirmed by southern blot analysis and/or by taqman copy number assay.longitudinal sections of the dead newborn mice revealed that the spine in the m-p49tg newborn was shifted around to the abdominal cavity from the back to the front .
cross sections showed that the thoracic cavity was asymmetric and the left side of the chest was smaller than that of the right .
both the left and right lungs were unexpanded in the m-p49tg mice that were dead at birth, and the heart morphology was substantially abnormal, without an appropriate division and/or formation of separate cardiac chambers in the m-p49tg mice vs. non-tg .
in the malformed m-p49tg mice that were dead at birth, the p49/strap expression was increased, while srf, anf, cardiac actin, α-mhc and β-mhc were decreased, in the whole body preparation, indicating that p49/strap overexpression altered the expression of these cardiac genes that are important in cardiac development and cellular cytoskeletal integrity  the cmv promoter was used to drive the expression of p49/strap transgene expression, the possibility of cmv promoter silencing was considered to be the possible mechanism in the normal-appearing mice without elevated p49/strap expression.
a number of reports indicate that cmv silencing can be reactivated by sodium butyrate  <cit> .
therefore, we sought to test this possibility in aortic smooth muscle  cells that were isolated from  <dig> month old tg and ntg mice.
p49/strap mrna expression was measured in response to sodium butyrate treatment.
as shown in figure 7b, p49/strap mrna level was slightly increased after the tg asm cells were treated for  <dig> hours in the presence of  <dig>  mm sodium butyrate .
however, p49/strap mrna level was increased significantly after the tg asm cells were treated for  <dig> hours in the presence of  <dig> mm sodium butyrate .
these findings indicate that in the present study, cmv promoter silencing may well have repressed p49/strap expression in those na-p49tg group, which were without abnormal phenotype and without increased p49/strap gene expression.
discussion
in the present study, we found that the p49/strap protein has a srf-binding domain at the n-terminus and a bud <dig> domain at the c-terminus.
the n-terminal domain containing the srf-binding domain was highly conserved among mammalian species, xenopus and zebrafish.
the c-terminal domain containing the bud <dig> domain was conserved not only among mammalian p49/strap proteins, but also conserved among yeast cellular morphogenesis proteins that regulate cell morphology and polarity.
in addition, overexpression of p49/strap in vitro and in vivo indeed changed the cell actin content, cell size and cell polarity.
p49/strap is one of the srf-binding proteins/cofactors that participate in the regulation of cytoskeletal and muscle-specific genes  <cit> .
the srf protein is a major transcription factor in regulating cardiac, skeletal and smooth muscle genes  <cit> .
srf has a dna binding domain at the n-terminus that binds to the sre  dna sequence, and a transcriptional activation domain at the c-terminus that is required for serum induction  <cit> .
srf and its cofactors usually form a protein complex which binds to srf target gene promoters and consequently activates or represses the target genes  <cit> .
therefore, srf cofactors play an important role in assisting srf to conduct tissue-specific and development stage-specific regulation of its target genes  <cit> .
among srf cofactors, the tcf cofactor, myocardin and mrtfs bind to the srf n-terminus, while p49/strap protein has been shown to bind to the srf transcriptional activation domain  <cit> .
it has been shown that the p49/strap protein is usually located within a protein complex containing the srf and myocardin proteins.
therefore, p49/strap is able to modulate the srf transcriptional activation  <cit> .
the localization of p49/strap protein has been studied experimentally, either through transfection assays or proteomic analyses  <cit> .
for instance, lamond’s group has reported that p49/strap   is among a number of proteins, including werner syndrome helicase, bloom’s syndrome helicase and nucleolin, that are localized in the nucleolus  <cit> .
recently, tafforeau et al.
demonstrated that p49/strap  is in the nucleolus and serves as a pre-rrna processing factor which regulates ribosome biogenesis  <cit> .
the authors stated that p49/strap did not have a yeast homologue  <cit> .
however, we have now observed that p49/strap does in fact, have a yeast homologue, namely bud <dig> based on the domain search in ncbi, pfam and interpro databases.
in addition, we observed that p49/strap is localized in both the nucleus and the nucleolus, which is similar to what has been reported for bud <dig>  bud <dig> is one of the proteins that regulate yeast bud site selection and polarized cell growth in response to both external and internal stimuli  <cit> .
polarized cell growth is a phenomenon that occurs in virtually all cells and it is essential for many important cellular events such as multicellular organ development and cell movement.
in addition, cell size and morphology are impacted by ribosome biogenesis which affects growth rate and cell size  <cit> .
cell morphology is also regulated by various bud proteins.
it has been shown that bud <dig> is involved in ribosome biogenesis, which affects both cell growth rate and cell size  <cit> .
therefore, it is likely that p49/strap may regulate cell size and morphology through its partnership with srf in regulating cytoskeletal genes, and may also regulate cell morphology and polarity with its bud <dig> domain.
cytoskeletal and muscle-specific genes are key components of cell morphology.
many of these genes are regulated by srf and its cofactors.
in the present study, p49/strap has been found to reduce actin fiber content and change cell morphology in cultured cells.
interestingly, the effect of p49/strap transfection on reducing cellular actin correlated with reduced actin expression in the malformed p49tg newborns.
the widespread structural and especially cardiac abnormalities in the dead newborns could be the consequence of altered expression of important cytoskeletal genes such as actin and other cardiac muscle-specific genes which include α-mhc and β-mhc, anf and srf itself.
the p49/strap protein, as a co-factor of srf, might be involved in the regulation of cytoskeletal and muscle-specific genes through potentially two different but related mechanisms.
first, p49/strap may interact with srf protein in the promoter of various cytoskeletal and muscle-specific genes and modulate their expression; second, p49/strap may indirectly modulate cytoskeletal and muscle-specific genes via regulating the expression of the srf gene itself, which is a major regulator of many other srf-target genes  <cit> .
the altered cellular actin dynamics may also indirectly regulate srf activation  <cit> .
in the transgenic mouse lines which were created in the present study, the p49/strap transgene expression was under the control of a cmv promoter, which is widely used to drive the ubiquitous expression of a transgene.
since the transgene overexpression can sometimes cause artifacts such as transcriptional squelching and may change balanced gene dosage and affect downstream regulation, it is also possible that the experimental results may not reflect the real function of a transgene.
this could happen when the overexpression of a transgene is very high  <cit> .
however, in the present study, all three p49tg lines each has only one transgene copy, and none of them had high transgene expression level.
therefore, the phenotypes observed in the present study are most likely to be the result of p49/strap overexpression instead of artifacts.
the p49tg mice appeared to separate into two different groups, those with severe defects that died at birth, and those without apparent defects/abnormality at birth and continued to live in the absence of noticeable phenotype.
the main difference between these two groups of p49tg offspring in terms of gene expression was in the level of p49/strap transgene expression: the p49tg with anatomical defects had elevated p49/strap gene expression, while the p49tg mice without defects had no significant elevation in p49/strap expression and their levels were similar to that of non-tg littermates.
these findings suggest that p49/strap overexpression clearly plays a critical role in the manifestation of the abnormal phenotype observed in the p49tg mice.
the finding that sodium butyrate-induced reactivation of p49/strap transgene mrna expression occurred in isolated aortic smooth muscle cells of the na-p49tg mice supports the notion that cmv promoter silencing most likely contributed to the repression of p49tg transgene expression in the subset of the p49tg mice that were without elevated p49/strap expression and without abnormal phenotype  <cit> .
the cmv promoter has been reported to have one cpg island with  <dig> cpg sites, which are subjected to dna methylation or hypermethylation.
consequently, the cmv promoter is sometimes repressed by dna methylation, which results in the decline, or complete silencing of the transgene expression  <cit> .
the cmv promoter that was used in the present p49tg study is  <dig> bp long, which is part of the pcdna3-p49/strap expression plasmid  <cit> .
the web-based software “cpgplot” was used to scan the sequence  <cit> , and a cpg island was found in the cmv promoter that could potentially be methylated , which may have silenced the p49/strap transgene in some mice.
cmv promoter methylation could occur both in vitro and in vivo  <cit> .
the degree of repression may vary from mouse to mouse, and may even be different among organs within a given animal  <cit> .
furthermore, the degree of cmv promoter repression could be related to the length of the promoter and the gc content in the cmv promoter sequence which is used in the given dna construct; it is also related to the transgene integration site  <cit> .
in addition to dna methylation, which may have occurred in the cmv promoter, it is known that histone deacetylation may also affect the cmv promoter activation  <cit> .
previously, we observed that overexpression of p49/strap itself altered the intracellular level of nad, reduced the nad/nadh ratio, and also induced the deacetylation of the srf protein  <cit> .
it is therefore plausible that overexpression of p49/strap itself could also change the acetylation status of other proteins, such as histones, and/or cause other epigenetic changes, including cmv promoter silencing.
epigenetic stability is important for the oocyte-to-embryo transition.
disruption of epigenetic stability could result in embryos displaying a large variety of phenotypes and growth defects during mouse embryogenesis  <cit> ; perhaps similar to what was observed in some of the malformed p49tg mice with elevated p49/strap expression.
similar to the reversal of other epigenetic modifications, dna methylation at the cmv promoter could also be reversed by means of pharmacological intervention.
it is plausible that reversing cmv methylation could potentially reactivate the transgene expression  <cit> .
future studies to test these possibilities will be of interest.
CONCLUSIONS
taken together, our findings indicate that p49/strap participates in the regulation of cytoskeletal gene expression and in the regulation of cell size as well as morphology/polarity.
elevated expression of p49/strap in p49tg mice resulted in various organ defects, including asymmetric  distribution of the vertebral spine and organs with altered expression of cardiac and skeletal muscle genes.
inasmuch as p49/strap is a srf binding protein, and we have observed that it is also a homolog of the yeast bud <dig> protein , it is plausible that p49/strap may serve as a bridge that links two important pathways: the srf-related pathway in transcriptional regulation of cytoskeletal genes and cell size , and the bud22-related pathway that regulates cell size, cell morphology and polarity.
we propose that the increased expression of p49/strap that is observed in aged human and rodent cells may mediate some of the observed changes in cellular morphology and the appearance of some of the markers of cellular senescence, including changes in actin structure and other intracellular-cytoskeletal proteins.
future studies in this area will likely yield substantial information.

 the protein p49/strap was identified in our laboratory in search of proteins that regulate cytoskeletal genes related to cardiac function. our initial observations indicated that p49/strap is a cofactor of serum response factor, contributing to the regulation of srf target genes. this gene was later named by the human genome organization as srfbp based on our initial finding that p49/strap binds to srf. serum response factor is an important transcription factor that regulates a number of immediate-early genes, cytoskeletal and muscle-specific genes. srf has been reported to exhibit functional interactions with a number of srf cofactors and/or binding proteins in the regulation of srf target genes. these interactions likely modulate srf function and may also enable srf to mediate tissue-specific regulation of its target genes at different developmental stages. a model of transcriptional regulation by srf and its binding proteins has been proposed, in which the interaction of multiple proteins affects the outcome of the expression of srf target genes. srf has two functional domains. the n-terminus is a dna binding domain that binds to the serum response element or carg box and the c-terminus is a transactivation domain. the p49/strap protein interacts directly with the srf transcriptional activation domain. therefore, the level of p49/strap expression and its affinity to srf protein may have a significant impact on srf activation. the p49/strap mrna and protein are expressed in multiple tissues, including heart, brain, liver, skeletal muscle and kidney. an increase of p49/strap expression has been observed in the myocardium of adult human and mouse with advancing age, and also in transgenic mouse models of cardiomyopathy. however, the functional domains of the p49/strap protein and the effect of increased in-vitro and in-vivo p49/strap expression remain to be defined. in the present study, the p49/strap protein sequence was analyzed for conserved domains. a srf-binding domain was found at the n-terminus of the p49/strap proteins of mammals and vertebrates; and a bud domain was found at the c-terminus of the p49/strap proteins and the yeast cellular morphogenesis protein. when p49/strap gene was overexpressed in cultured cells and in transgenic mice, morphological alterations were observed, both at the cellular and whole organ, structural level.  bioinformatics analysis of p49/strap protein the p49/strap protein sequences of human, mouse and rat were analyzed using bioinformatics tools. sequence alignment revealed that there was approximately 60% homology among human, mouse and rat p49/strap proteins. further sequence alignment of mammalian p49/strap proteins, including that of chimpanzee and dog, showed a 52% homology in p49/strap proteins. two conserved regions were found at the n-terminus and c-terminus. the conserved “n-region” overlaps with the “srf-binding domain” that was found to interact with srf in our previous study. the p49/strap protein sequences were compared against the “conserved domain database” at the national center for biotechnology information, the “pfam” and the interpro databases. the c-region of p49/strap matched an existing conserved domain in all three databases, specifically the bud domain of the cellular morphogenesis protein that regulates yeast budding selection, polarity and rna biogenesis. each database showed a slightly different length of bud domain in p49/strap protein. as for the human p49/strap, the bud domain was at amino acid 399– in the ncbi conserved domain database; amino acid 329– in the pfam database, and amino acid 360– in the interpro database, respectively. alignment of the bud sequence of yeast strands revealed that there was a conserved domain at the c-terminus, ranging from amino acid to the end of the sequences. when the p49/strap protein sequences of mammalian species were compared with the yeast bud proteins, it was found that only part of sequences at the end of c-region, approximately amino acids, was homologous among the sequences, indicating that those sequences represented a highly conserved region in the bud domain. bioinformatics analysis indicated that there are four nls sequences in the human, mouse and rat p49/strap proteins, while three nls sequences were found within bud of s. cerevisiae and four nlss in bud of s. pombe. it was also predicted that mouse p49/strap had two nucleolar localization sequences, and baker’s yeast bud had three nucleolar localization sequences. in addition, it was also predicted that both p49/strap and bud proteins would be present in the cytoplasmic compartments, including mitochondria, endoplasmic reticulum, plasma membrane and vesicles of the secretory system. both p and bud proteins were predicted to be present in the nucleus, but also in the cytoplasmic compartments, including mitochondria, endoplasmic reticulum, plasma membrane and vesicles of the secretory system. intracellular distribution of p49/strap we previously observed that transfected gfp-p49/strap fusion protein was mainly localized in the nucleus of nih3t cells, where it acts as a srf cofactor to regulate srf-target genes. indeed, bioinformatics analysis revealed that the mouse p49/strap had both nuclear localization signal sequences and nucleolar localization signal sequences.to confirm the bioinformatics result, h9c cells were transfected with the gfp-expression control plasmid and gfp-p49/strap expression plasmid, respectively. as shown in figure 2a and c, the green gfp fluorescence was present in the entire cell in gfp-control plasmid transfected cell. by contrast, in the gfp-p49/strap expression plasmid transfected cells, the gfp-p49/strap protein appeared to be present mainly in nucleus, with a few very bright dots within the nucleus. we used nucleolin to co-stain the transfected cells. as shown in figure 2e, nucleolin was stained in red. when figure 2d and e were merged, the bright green-colored gfp-p49/strap dots co-localized with the red-colored nucleolin, indicating that the p49/strap protein does localize within the nucleolus. the p49/strap protein is known to be highly expressed in multiple tissues based on western blotting. to better understand the intracellular distribution of the endogenous p49/strap protein, an affinity-purified antibody against a p49/strap epitope was used for immunochemistry staining of h9c cells. as shown in figure 2g, the endogenous p49/strap protein was present in both the nucleus and cytoplasm. in addition, the p49/strap protein was widely distributed within the cell in close proximity to the actin fibers; some of the p49/strap proteins appeared to form a “tail” that was co-located at the end of the actin fibers, close to their attachment sites to the inner wall of the cell membrane. these findings suggest that p49/strap may play a role in cellular cytoskeletal structure and function via its presence in the cytoplasm. p49/strap overexpression affects cell morphology to determine the effect of p49/strap overexpression on cellular morphology, we infected h9c cells with the gfp-control adenovirus or recombinant p49/strap adenovirus. at hours post-infection, the cells were fixed and immunocytochemistry staining was performed. the phalloidin was used to stain f-actin. the p49/strap antibody and alexa fluor labelled secondary antibody were used to stain the cells to visualize the fluorescent intensity of both the endogenous and the exogenous p49/strap proteins in the p49/strap-virus treated cells versus that of the endogenous p49/strap protein only in the control cells infected with gfp-control virus.as shown in figure 3a-d, in the gfp-control virus infected cells, the actin fibers were present as thick, well aligned bundles, and the p protein was mainly localized in the nucleus; however, in the p49/strap virus treated cells, the structure of the actin fibers was more diffuse, especially in the perinuclear region, and the intensity of the actin fibers was weaker. asymmetric distribution of the p49/strap protein was observed within the p49/strap treated cells compared to that in the control cells. cell size measurements revealed that h9c cells treated with the p49/strap adenovirus were smaller compared to that of the control adenovirus infected cells. in addition, p49/strap treated cells showed much larger nucleocytoplasmic ratios compared to that of the control, 1:  ±  versus 1:  ± , respectively. the overall microscopic observation revealed that increased p49/strap protein reduced the f-actin structure and intensity, and changed the cell morphology in term of cytoskeletal structure, cell size, cell shape and nucleocytoplasmic ratio. p49/strap modulated srf gene expression p49/strap is a srf cofactor that co-regulates srf-target genes. since there are two srf-binding sites in the srf gene promoter, the srf gene itself is also a srf-target gene subject to the regulation by srf. to test the hypothesis that p49/strap may regulate the srf gene, a plasmid containing firefly luciferase driven by the srf gene promoter was used in the luciferase-based promoter activity assay. as shown in figure 5a, the srf plasmid transfection induced srf gene promoter activity; when p49/strap was co-transfected with srf, p49/strap repressed the srf-induced promoter activity. another srf cofactor, myocardin was also used in the transfection assay; myocardin overexpression induced srf gene promoter activity by approximately 5-fold, while p49/strap repressed the srf-promoter activity that was induced by myocardin plasmid transfection. the mrna levels of srf and atrial natriuretic peptide genes were also measured in response to p49/strap overexpression. as shown in figure 5b, p49/strap repressed the expression of both the srf gene and the anf gene. generation of p49/strap transgenic mouse lines the p49/strap mrna/protein is well expressed in multiple tissues, including brain, heart, skeletal muscle, kidney, liver and testis. to test the hypothesis that increased expression of p49/strap in vivo may alter the expression of srf-target genes and affect the morphology of mouse cells and tissues, we generated transgenic mouse lines containing a dna construct with p49/strap cdna under the transcriptional control of the cmv promoter. after multiple microinjections and screenings, three transgenic founder mice were identified. the founder mice were bred to non-transgenic mice, and three p49tg lines were thus established. both southern blotting and quantitative pcr analysis were used for the detection of the transgene copy number of the three p49tg lines. each line was found to have only one transgene copy. death of some p49/strap transgenic newborn mice the p49tg founder mice that survived to adulthood were fertile without apparent abnormality in reproduction. the p49tg offspring had their genotype confirmed by pcr. an examination of litters from generations of the p49tg mice revealed that the transgenic offspring separated into two groups based on p49/strap mrna expression level and their phenotype. a close check of three mouse generations revealed that among newborn p49tg mice, newborns were malformed, which accounted for 43% of the p49tg offspring. the malformed group of newborn p49tg mice displayed serious defects and almost all of them were either dead at birth or died immediately thereafter. these malformed mice had elevated level of p49/strap mrna expression. the other group of newborn p49tg mice were grossly normal-appearing, had no apparent anatomic defects and most of these p49tg mice survived without visible abnormality. these na-p49tg mice had similar levels of p49/strap mrna expression compared with that of the non-tg mice. however, their p49/strap transgenic status was confirmed by southern blot analysis and/or by taqman copy number assay.longitudinal sections of the dead newborn mice revealed that the spine in the m-p49tg newborn was shifted around to the abdominal cavity from the back to the front. cross sections showed that the thoracic cavity was asymmetric and the left side of the chest was smaller than that of the right. both the left and right lungs were unexpanded in the m-p49tg mice that were dead at birth, and the heart morphology was substantially abnormal, without an appropriate division and/or formation of separate cardiac chambers in the m-p49tg mice vs. non-tg. in the malformed m-p49tg mice that were dead at birth, the p49/strap expression was increased, while srf, anf, cardiac actin, α-mhc and β-mhc were decreased, in the whole body preparation, indicating that p49/strap overexpression altered the expression of these cardiac genes that are important in cardiac development and cellular cytoskeletal integrity the cmv promoter was used to drive the expression of p49/strap transgene expression, the possibility of cmv promoter silencing was considered to be the possible mechanism in the normal-appearing mice without elevated p49/strap expression. a number of reports indicate that cmv silencing can be reactivated by sodium butyrate. therefore, we sought to test this possibility in aortic smooth muscle cells that were isolated from month old tg and ntg mice. p49/strap mrna expression was measured in response to sodium butyrate treatment. as shown in figure 7b, p49/strap mrna level was slightly increased after the tg asm cells were treated for hours in the presence of mm sodium butyrate. however, p49/strap mrna level was increased significantly after the tg asm cells were treated for hours in the presence of mm sodium butyrate. these findings indicate that in the present study, cmv promoter silencing may well have repressed p49/strap expression in those na-p49tg group, which were without abnormal phenotype and without increased p49/strap gene expression. discussion in the present study, we found that the p49/strap protein has a srf-binding domain at the n-terminus and a bud domain at the c-terminus. the n-terminal domain containing the srf-binding domain was highly conserved among mammalian species, xenopus and zebrafish. the c-terminal domain containing the bud domain was conserved not only among mammalian p49/strap proteins, but also conserved among yeast cellular morphogenesis proteins that regulate cell morphology and polarity. in addition, overexpression of p49/strap in vitro and in vivo indeed changed the cell actin content, cell size and cell polarity. p49/strap is one of the srf-binding proteins/cofactors that participate in the regulation of cytoskeletal and muscle-specific genes. the srf protein is a major transcription factor in regulating cardiac, skeletal and smooth muscle genes. srf has a dna binding domain at the n-terminus that binds to the sre dna sequence, and a transcriptional activation domain at the c-terminus that is required for serum induction. srf and its cofactors usually form a protein complex which binds to srf target gene promoters and consequently activates or represses the target genes. therefore, srf cofactors play an important role in assisting srf to conduct tissue-specific and development stage-specific regulation of its target genes. among srf cofactors, the tcf cofactor, myocardin and mrtfs bind to the srf n-terminus, while p49/strap protein has been shown to bind to the srf transcriptional activation domain. it has been shown that the p49/strap protein is usually located within a protein complex containing the srf and myocardin proteins. therefore, p49/strap is able to modulate the srf transcriptional activation. the localization of p49/strap protein has been studied experimentally, either through transfection assays or proteomic analyses. for instance, lamond’s group has reported that p49/strap is among a number of proteins, including werner syndrome helicase, bloom’s syndrome helicase and nucleolin, that are localized in the nucleolus. recently, tafforeau et al. demonstrated that p49/strap is in the nucleolus and serves as a pre-rrna processing factor which regulates ribosome biogenesis. the authors stated that p49/strap did not have a yeast homologue. however, we have now observed that p49/strap does in fact, have a yeast homologue, namely bud based on the domain search in ncbi, pfam and interpro databases. in addition, we observed that p49/strap is localized in both the nucleus and the nucleolus, which is similar to what has been reported for bud bud is one of the proteins that regulate yeast bud site selection and polarized cell growth in response to both external and internal stimuli. polarized cell growth is a phenomenon that occurs in virtually all cells and it is essential for many important cellular events such as multicellular organ development and cell movement. in addition, cell size and morphology are impacted by ribosome biogenesis which affects growth rate and cell size. cell morphology is also regulated by various bud proteins. it has been shown that bud is involved in ribosome biogenesis, which affects both cell growth rate and cell size. therefore, it is likely that p49/strap may regulate cell size and morphology through its partnership with srf in regulating cytoskeletal genes, and may also regulate cell morphology and polarity with its bud domain. cytoskeletal and muscle-specific genes are key components of cell morphology. many of these genes are regulated by srf and its cofactors. in the present study, p49/strap has been found to reduce actin fiber content and change cell morphology in cultured cells. interestingly, the effect of p49/strap transfection on reducing cellular actin correlated with reduced actin expression in the malformed p49tg newborns. the widespread structural and especially cardiac abnormalities in the dead newborns could be the consequence of altered expression of important cytoskeletal genes such as actin and other cardiac muscle-specific genes which include α-mhc and β-mhc, anf and srf itself. the p49/strap protein, as a co-factor of srf, might be involved in the regulation of cytoskeletal and muscle-specific genes through potentially two different but related mechanisms. first, p49/strap may interact with srf protein in the promoter of various cytoskeletal and muscle-specific genes and modulate their expression; second, p49/strap may indirectly modulate cytoskeletal and muscle-specific genes via regulating the expression of the srf gene itself, which is a major regulator of many other srf-target genes. the altered cellular actin dynamics may also indirectly regulate srf activation. in the transgenic mouse lines which were created in the present study, the p49/strap transgene expression was under the control of a cmv promoter, which is widely used to drive the ubiquitous expression of a transgene. since the transgene overexpression can sometimes cause artifacts such as transcriptional squelching and may change balanced gene dosage and affect downstream regulation, it is also possible that the experimental results may not reflect the real function of a transgene. this could happen when the overexpression of a transgene is very high. however, in the present study, all three p49tg lines each has only one transgene copy, and none of them had high transgene expression level. therefore, the phenotypes observed in the present study are most likely to be the result of p49/strap overexpression instead of artifacts. the p49tg mice appeared to separate into two different groups, those with severe defects that died at birth, and those without apparent defects/abnormality at birth and continued to live in the absence of noticeable phenotype. the main difference between these two groups of p49tg offspring in terms of gene expression was in the level of p49/strap transgene expression: the p49tg with anatomical defects had elevated p49/strap gene expression, while the p49tg mice without defects had no significant elevation in p49/strap expression and their levels were similar to that of non-tg littermates. these findings suggest that p49/strap overexpression clearly plays a critical role in the manifestation of the abnormal phenotype observed in the p49tg mice. the finding that sodium butyrate-induced reactivation of p49/strap transgene mrna expression occurred in isolated aortic smooth muscle cells of the na-p49tg mice supports the notion that cmv promoter silencing most likely contributed to the repression of p49tg transgene expression in the subset of the p49tg mice that were without elevated p49/strap expression and without abnormal phenotype. the cmv promoter has been reported to have one cpg island with cpg sites, which are subjected to dna methylation or hypermethylation. consequently, the cmv promoter is sometimes repressed by dna methylation, which results in the decline, or complete silencing of the transgene expression. the cmv promoter that was used in the present p49tg study is bp long, which is part of the pcdna3-p49/strap expression plasmid. the web-based software “cpgplot” was used to scan the sequence, and a cpg island was found in the cmv promoter that could potentially be methylated, which may have silenced the p49/strap transgene in some mice. cmv promoter methylation could occur both in vitro and in vivo. the degree of repression may vary from mouse to mouse, and may even be different among organs within a given animal. furthermore, the degree of cmv promoter repression could be related to the length of the promoter and the gc content in the cmv promoter sequence which is used in the given dna construct; it is also related to the transgene integration site. in addition to dna methylation, which may have occurred in the cmv promoter, it is known that histone deacetylation may also affect the cmv promoter activation. previously, we observed that overexpression of p49/strap itself altered the intracellular level of nad, reduced the nad/nadh ratio, and also induced the deacetylation of the srf protein. it is therefore plausible that overexpression of p49/strap itself could also change the acetylation status of other proteins, such as histones, and/or cause other epigenetic changes, including cmv promoter silencing. epigenetic stability is important for the oocyte-to-embryo transition. disruption of epigenetic stability could result in embryos displaying a large variety of phenotypes and growth defects during mouse embryogenesis; perhaps similar to what was observed in some of the malformed p49tg mice with elevated p49/strap expression. similar to the reversal of other epigenetic modifications, dna methylation at the cmv promoter could also be reversed by means of pharmacological intervention. it is plausible that reversing cmv methylation could potentially reactivate the transgene expression. future studies to test these possibilities will be of interest.  taken together, our findings indicate that p49/strap participates in the regulation of cytoskeletal gene expression and in the regulation of cell size as well as morphology/polarity. elevated expression of p49/strap in p49tg mice resulted in various organ defects, including asymmetric distribution of the vertebral spine and organs with altered expression of cardiac and skeletal muscle genes. inasmuch as p49/strap is a srf binding protein, and we have observed that it is also a homolog of the yeast bud protein, it is plausible that p49/strap may serve as a bridge that links two important pathways: the srf-related pathway in transcriptional regulation of cytoskeletal genes and cell size, and the bud22-related pathway that regulates cell size, cell morphology and polarity. we propose that the increased expression of p49/strap that is observed in aged human and rodent cells may mediate some of the observed changes in cellular morphology and the appearance of some of the markers of cellular senescence, including changes in actin structure and other intracellular-cytoskeletal proteins. future studies in this area will likely yield substantial information. 
transfected gfp-p49/strap protein co-localized with nucleolin within the nucleolus. the protein p49/strap is a transcription cofactor of serum response factor which regulates cytoskeletal and muscle-specific genes. the bud domain was conserved among mammalian p49/strap proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. overexpression of p49/strap reduced actin content in cultured cells and resulted in smaller cell size versus control cells. since p49/strap is a co-factor of srf, our data suggest that p49/strap likely regulates cell size and morphology through srf target genes. p49/strap altered the expression of certain muscle-specific genes, including that of the srf gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two sre binding sites. increased expression of p49/strap in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. transcription cofactorhomologysubcellular localizationcell morphologytransgenic micecmv promoter reactivation two conserved domains were found in the p49/strap protein. the observed increase in p49/strap expression during cellular aging may contribute to observed morphological changes in senescence. the srf-binding domain was at its n-terminus and was highly conserved among mammalian species, xenopus and zebrafish.
 the protein p49/strap is a transcription cofactor of serum response factor which regulates cytoskeletal and muscle-specific genes.  two conserved domains were found in the p49/strap protein. the srf-binding domain was at its n-terminus and was highly conserved among mammalian species, xenopus and zebrafish. a bud domain was found at its c-terminus in three sequence databases. the bud domain was conserved among mammalian p49/strap proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. the endogenous p49/srap protein was localized mainly in the nucleus but also widely distributed in the cytoplasm, and was in close proximity to the actin. transfected gfp-p49/strap protein co-localized with nucleolin within the nucleolus. overexpression of p49/strap reduced actin content in cultured cells and resulted in smaller cell size versus control cells. increased expression of p49/strap in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. p49/strap altered the expression of certain muscle-specific genes, including that of the srf gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two sre binding sites.  since p49/strap is a co-factor of srf, our data suggest that p49/strap likely regulates cell size and morphology through srf target genes. the function of its bud domain warrants further investigation. the observed increase in p49/strap expression during cellular aging may contribute to observed morphological changes in senescence. transcription cofactorhomologysubcellular localizationcell morphologytransgenic micecmv promoter reactivation 