Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse.

KEY POINTS: Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix. Deletion of the desmin gene in mdx mice [double knockout (DKO) mice] induces marked muscle weakness and fatigue resistance compared to mdx mice. Muscle fragility (higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice. By contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy. Desmin cDNA transfer with adeno-associated virus in newborn mdx mice reduced muscle weakness. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic muscle. ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by dystrophin deficiency. Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix that contributes to muscle function. In the present study, we attempted to provide further insight into the roles of desmin, for which the expression is increased in the muscle from the mouse mdx DMD model. We show that a deletion of the desmin gene (Des) in mdx mice [double knockout (DKO) mice, mdx:desmin-/-] induces a marked muscle weakness; namely, a reduced absolute maximal force production and increased fatigue compared to that in mdx mice. Fragility (i.e. higher susceptibility to contraction-induced injury) was also aggravated in DKO mice compared to mdx mice, despite the promotion of supposedly less fragile muscle fibres in DKO mice, and this worsening of fragility was related to a decreased muscle excitability. Moreover, in contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy, as indicated by smaller and fewer fibres, with a reduced percentage of centronucleated fibres, potentially explaining the severe muscle weakness. Notably, Desmin cDNA transfer with adeno-associated virus in newborn mdx mice improved specific maximal force normalized to muscle weight. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic mdx mice, which differ, at least in part, from those observed in healthy muscle.

Synemin acts as a regulator of signalling molecules during skeletal muscle hypertrophy.

Synemin, a type IV intermediate filament (IF) protein, forms a bridge between IFs and cellular membranes. As an A-kinase-anchoring protein, it also provides temporal and spatial targeting of protein kinase A (PKA). However, little is known about its functional roles in either process. To better understand its functions in muscle tissue, we generated synemin-deficient (Synm(-) (/-)) mice. Synm(-) (/-) mice displayed normal development and fertility but showed a mild degeneration and regeneration phenotype in myofibres and defects in sarcolemma membranes. Following mechanical overload, Synm(-) (/-) mice muscles showed a higher hypertrophic capacity with increased maximal force and fatigue resistance compared with control mice. At the molecular level, increased remodelling capacity was accompanied by decreased myostatin (also known as GDF8) and atrogin (also known as FBXO32) expression, and increased follistatin expression. Furthermore, the activity of muscle-mass control molecules (the PKA RIIα subunit, p70S6K and CREB1) was increased in mutant mice. Finally, analysis of muscle satellite cell behaviour suggested that the absence of synemin could affect the balance between self-renewal and differentiation of these cells. Taken together, our results show that synemin is necessary to maintain membrane integrity and regulates signalling molecules during muscle hypertrophy.

SRF selectively controls tip cell invasive behavior in angiogenesis.

Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Mechanical Overloading Increases Maximal Force and Reduces Fragility in Hind Limb Skeletal Muscle from Mdx Mouse.

There is fear that mechanical overloading (OVL; ie, high-force contractions) accelerates Duchenne muscular dystrophy. Herein, we determined whether short-term OVL combined with wheel running, short-term OVL combined with irradiation, and long-term OVL are detrimental for hind limb mdx mouse muscle, a murine model of Duchene muscular dystrophy exhibiting milder dystrophic features. OVL was induced by the surgical ablation of the synergic muscles of the plantaris muscle, a fast muscle susceptible to contraction-induced muscle damage in mdx mice. We found that short-term OVL combined with wheel and long-term OVL did not worsen the deficit in specific maximal force (ie, absolute maximal force normalized to muscle size) and histological markers of muscle damage (percentage of regenerating fibers and fibrosis) in mdx mice. Moreover, long-term OVL did not increase the alteration in calcium homeostasis and did not deplete muscle cell progenitors expressing Pax 7 in mdx mice. Irradiation before short-term OVL, which is believed to inhibit muscle regeneration, was not more detrimental to mdx than control mice. Interestingly, short-term OVL combined with wheel and long-term OVL markedly improved the susceptibility to contraction-induced damage, increased absolute maximal force, induced hypertrophy, and promoted a slower, more oxidative phenotype. Together, these findings indicate that OVL is beneficial to mdx muscle, and muscle regeneration does not mask the potentially detrimental effect of OVL.

CTIP2 is a negative regulator of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is involved in physiological and pathological events including inflammation, cancer, AIDS, and cardiac hypertrophy. The balance between its active and inactive form is tightly controlled to ensure cellular integrity. We report that the transcriptional repressor CTIP2 is a major modulator of P-TEFb activity. CTIP2 copurifies and interacts with an inactive P-TEFb complex containing the 7SK snRNA and HEXIM1. CTIP2 associates directly with HEXIM1 and, via the loop 2 of the 7SK snRNA, with P-TEFb. In this nucleoprotein complex, CTIP2 significantly represses the Cdk9 kinase activity of P-TEFb. Accordingly, we show that CTIP2 inhibits large sets of P-TEFb- and 7SK snRNA-sensitive genes. In hearts of hypertrophic cardiomyopathic mice, CTIP2 controls P-TEFb-sensitive pathways involved in the establishment of this pathology. Overexpression of the ß-myosin heavy chain protein contributes to the pathological cardiac wall thickening. The inactive P-TEFb complex associates with CTIP2 at the MYH7 gene promoter to repress its activity. Taken together, our results strongly suggest that CTIP2 controls P-TEFb function in physiological and pathological conditions.

Endothelial mineralocorticoid receptor activation enhances endothelial protein C receptor and decreases vascular thrombosis in mice.

Previous studies have shown that aldosterone, which activates the mineralocorticoid receptor (MR), promotes thrombosis in animal models. Our objective was to determine whether MR activation/expression in the vascular endothelium could modify thrombotic risk in vivo and to examine thrombin generation at the surface of aortic endothelial cells (HAECs). MR was conditionally overexpressed in vivo in vascular endothelial cells in mice (MR-EC mice) or stimulated with aldosterone in HAECs. Thrombosis after ferric chloride injury was delayed in MR-EC mice compared with controls as well as in wild-type FVB/NRj mice treated with aldosterone (60 µg/kg/d for 21 d). Thrombin generation in platelet-poor plasma did not differ between MR-EC mice and controls. In MR-EC mice, aortic endothelial cell protein C receptor (EPCR) expression was increased. Aldosterone (10(-8) M) attenuated thrombin generation at the surface of cultured HAECs, and this effect was associated with up-regulation of expression of EPCR, which promotes formation of activated protein C. Aldosterone increases EPCR expression via a transcriptional mechanism involving interaction of MR with the specificity protein 1 site. These findings demonstrate that MR activation acts on endothelial cells to protect against thrombosis in physiological conditions and that MR-mediated EPCR overexpression drives this antithrombotic property through enhancing protein C activation.

Generation of induced pluripotent stem cell line (TMOi001-A-11) carrying a homozygous deletion in the synemin gene using CRISPR/Cas9.

A number of genetic variants in the SYNM gene encoding for the intermediate filament synemin have been reported in patients with cardiomyopathies, skeletal myopathies, cancer and certain neurodegenerative disorders. To better understand its role, we generated a human induced pluripotent stem cell line with a homozygous deletion in the SYNM gene by CRISPR/Cas9 genome editing. The synemin-knockout human induced pluripotent stem cells exhibit typical morphology of pluripotent cells, expression of pluripotency markers, normal karyotype and differentiation capacity in the three germ layers. This line will allow us to investigate the role of synemin in cardiomyopathy upon differentiation into beating cardiomyocytes.

Smooth muscle αv integrins regulate vascular fibrosis via CD109 downregulation of TGF-ß signalling.

Aims: αv integrins are implicated in fibrosis in a number of organs through their ability to activate TGF-ß. However their role in vascular fibrosis and collagen accumulation is only partially understood. Here we have used αv conditional knockout mice and cell lines to determine how αv contributes to vascular smooth muscle cell (VSMC) function in vascular fibrosis and the role of TGF-ß in that process. Methods and results: Angiotensin II (Ang II) treatment causes upregulation of αv and ß3 expression in the vessel wall, associated with increased collagen deposition. We found that deletion of αv integrin subunit from VSMCs (αv SMKO) protected mice against angiotensin II-induced collagen production and assembly. Transcriptomic analysis of the vessel wall in αv SMKO mice and controls identified a significant reduction in expression of fibrosis and related genes in αv SMKO mice. In contrast, αv SMKO mice showed prolonged expression of CD109, which is known to affect TGF-ß signalling. Using cultured mouse and human VSMCs, we showed that overexpression of CD109 phenocopied knockdown of αv integrin, attenuating collagen expression, TGF-ß activation, and Smad2/3 signalling in response to angiotensin II or TGF-ß stimulation. CD109 and TGF-ß receptor were internalized in early endosomes. Conclusion: We identify a role for VSMC αv integrin in vascular fibrosis and show that αv acts in concert with CD109 to regulate TGF-ß signalling.

Absence of Desmin Results in Impaired Adaptive Response to Mechanical Overloading of Skeletal Muscle.

Background: Desmin is a muscle-specific protein belonging to the intermediate filament family. Desmin mutations are linked to skeletal muscle defects, including inherited myopathies with severe clinical manifestations. The aim of this study was to examine the role of desmin in skeletal muscle remodeling and performance gain induced by muscle mechanical overloading which mimics resistance training. Methods: Plantaris muscles were overloaded by surgical ablation of gastrocnemius and soleus muscles. The functional response of plantaris muscle to mechanical overloading in desmin-deficient mice (DesKO, n = 32) was compared to that of control mice (n = 36) after 7-days or 1-month overloading. To elucidate the molecular mechanisms implicated in the observed partial adaptive response of DesKO muscle, we examined the expression levels of genes involved in muscle growth, myogenesis, inflammation and oxidative energetic metabolism. Moreover, ultrastructure and the proteolysis pathway were explored. Results: Contrary to control, absolute maximal force did not increase in DesKO muscle following 1-month mechanical overloading. Fatigue resistance was also less increased in DesKO as compared to control muscle. Despite impaired functional adaptive response of DesKO mice to mechanical overloading, muscle weight and the number of oxidative MHC2a-positive fibers per cross-section similarly increased in both genotypes after 1-month overloading. However, mechanical overloading-elicited remodeling failed to activate a normal myogenic program after 7-days overloading, resulting in proportionally reduced activation and differentiation of muscle stem cells. Ultrastructural analysis of the plantaris muscle after 1-month overloading revealed muscle fiber damage in DesKO, as indicated by the loss of sarcomere integrity and mitochondrial abnormalities. Moreover, the observed accumulation of autophagosomes and lysosomes in DesKO muscle fibers could indicate a blockage of autophagy. To address this issue, two main proteolysis pathways, the ubiquitin-proteasome system and autophagy, were explored in DesKO and control muscle. Our results suggested an alteration of proteolysis pathways in DesKO muscle in response to mechanical overloading. Conclusion: Taken together, our results show that mechanical overloading increases the negative impact of the lack of desmin on myofibril organization and mitochondria. Furthermore, our results suggest that under these conditions, the repairing activity of autophagy is disturbed. Consequently, force generation is not improved despite muscle growth, suggesting that desmin is required for a complete response to resistance training in skeletal muscle.

Conditional inactivation of TGF-ß type II receptor in smooth muscle cells and epicardium causes lethal aortic and cardiac defects.

To understand the role of TGF-ß signaling in cardiovascular development, we generated mice with conditional deletion of the TGF-ß type II receptor (TßRII) gene (Tgfbr2) in cells expressing the smooth muscle cell-specific protein SM22α. The SM22α promoter was active in tissues involved in cardiovascular development: vascular smooth muscle cells (VSMCs), epicardium and myocardium. All SM22-Cre(+/-)/Tgfbr2 (flox/flox) embryos died during the last third of gestation. About half the mutant embryos exhibited heart defects (ventricular myocardium hypoplasia and septal defects). All mutant embryos displayed profound vascular abnormalities in the descending thoracic aorta (irregular outline and thickness, occasional aneurysms and elastic fiber disarray). Restriction of these defects to the descending thoracic aorta occurred despite similar levels of Tgfbr2 invalidation in the other portions of the aorta, the ductus arteriosus and the pulmonary trunk. Immunocytochemistry identified impairment of VSMC differentiation in the coronary vessels and the descending thoracic aorta as crucial for the defects. Ventricular myocardial hypoplasia, when present, was associated to impaired α-SMA differentiation of the epicardium-derived coronary VSMCs. Tgfbr2 deletion in the VSMCs of the descending thoracic aorta diminished the number of α-SMA-positive VSMC progenitors in the media at E11.5 and drastically decreased tropoelastin (from E11.5) and fibulin-5 (from E.12.5) synthesis and/or deposition. Defective elastogenesis observed in all mutant embryos and the resulting dilatation and probable rupture of the descending thoracic aorta might explain the late embryonic lethality. To conclude, during mouse development, TGF-ß plays an irreplaceable role on the differentiation of the VSMCs in the coronary vessels and the descending thoracic aorta.

Bcl11b/Ctip2 in Skin, Tooth, and Craniofacial System.

Ctip2/Bcl11b is a zinc finger transcription factor with dual action (repression/activation) that couples epigenetic regulation to gene transcription during the development of various tissues. It is involved in a variety of physiological responses under healthy and pathological conditions. Its role and mechanisms of action are best characterized in the immune and nervous systems. Furthermore, its implication in the development and homeostasis of other various tissues has also been reported. In the present review, we describe its role in skin development, adipogenesis, tooth formation and cranial suture ossification. Experimental data from several studies demonstrate the involvement of Bcl11b in the control of the balance between cell proliferation and differentiation during organ formation and repair, and more specifically in the context of stem cell self-renewal and fate determination. The impact of mutations in the coding sequences of Bcl11b on the development of diseases such as craniosynostosis is also presented. Finally, we discuss genome-wide association studies that suggest a potential influence of single nucleotide polymorphisms found in the 3' regulatory region of Bcl11b on the homeostasis of the cardiovascular system.

New role for serum response factor in postnatal skeletal muscle growth and regeneration via the interleukin 4 and insulin-like growth factor 1 pathways.

Serum response factor (SRF) is a crucial transcriptional factor for muscle-specific gene expression. We investigated SRF function in adult skeletal muscles, using mice with a postmitotic myofiber-targeted disruption of the SRF gene. Mutant mice displayed severe skeletal muscle mass reductions due to a postnatal muscle growth defect resulting in highly hypotrophic adult myofibers. SRF-depleted myofibers also failed to regenerate following injury. Muscles lacking SRF had very low levels of muscle creatine kinase and skeletal alpha-actin (SKA) transcripts and displayed other alterations to the gene expression program, indicating an overall immaturity of mutant muscles. This loss of SKA expression, together with a decrease in beta-tropomyosin expression, contributed to myofiber growth defects, as suggested by the extensive sarcomere disorganization found in mutant muscles. However, we observed a downregulation of interleukin 4 (IL-4) and insulin-like growth factor 1 (IGF-1) expression in mutant myofibers which could also account for their defective growth and regeneration. Indeed, our demonstration of SRF binding to interleukin 4 and IGF-1 promoters in vivo suggests a new crucial role for SRF in pathways involved in muscle growth and regeneration.

Mosaic inactivation of the serum response factor gene in the myocardium induces focal lesions and heart failure.

BACKGROUND AND AIMS: Regional alterations in ventricular mechanical functions are a primary determinant for the risk of myocardial injuries in various cardiomyopathies. The serum response factor (SRF) is a transcription factor regulating contractile and cytoskeletal genes and may play an important role in the remodelling of myocardium at the cellular level. METHODS: Using Desmin-Cre transgenic mice, we generated a model of mosaic inactivation of a floxed-Srf allele in the heart to analyze the consequence of regional alterations of SRF-mediated functions in the myocardium. RESULTS: Two types of cardiomyocytes co-existed in the Desmin-Cre:Sf/Sf mice. Cardiomyocytes lacking SRF became thin and elongated while cardiomyocytes containing SRF became hypertrophic. Several physiological contractile genes were down-regulated while skeletal alpha-actin was induced in SRF positive area only. Mutants developed heart failure associated with the presence of focal lesions in the myocardium, and died before month 11. CONCLUSIONS: Juxtaposition of functional SRF wild-type and failing SRF mutant cardiomyocytes generates deleterious heterogeneity in the myocardium. Our results show that SRF contributes to the capacity of cardiomyocytes to remodel their shape and contractile functions in response to their local environment; suggesting that it may play a role in pathologies involving regional alterations of ventricular mechanics in the heart.

Increasing autophagy does not affect neurogenic muscle atrophy.

Physiological autophagy plays a crucial role in the regulation of muscle mass and metabolism, while the excessive induction or the inhibition of the autophagic flux contributes to the progression of several diseases. Autophagy can be activated by different stimuli, including cancer, exercise, caloric restriction and denervation. The latter leads to muscle atrophy through the activation of catabolic pathways, i.e. the ubiquitin-proteasome system and autophagy. However, the kinetics of autophagy activation and the upstream molecular pathways in denervated skeletal muscle have not been reported yet. In this study, we characterized the kinetics of autophagic induction, quickly triggered by denervation, and report the Akt/mTOR axis activation. Besides, with the aim to assess the relative contribution of autophagy in neurogenic muscle atrophy, we triggered autophagy with different stimuli along with denervation, and observed that four week-long autophagic induction, by either intermitted fasting or rapamycin treatment, did not significantly affect muscle mass loss. We conclude that: i) autophagy does not play a major role in inducing muscle loss following denervation; ii) nonetheless, autophagy may have a regulatory role in denervation induced muscle atrophy, since it is significantly upregulated as early as eight hours after denervation; iii) Akt/mTOR axis, AMPK and FoxO3a are activated consistently with the progression of muscle atrophy, further highlighting the complexity of the signaling response to the atrophying stimulus deriving from denervation.

Publisher Correction: Vimentin knockout results in increased expression of sub-endothelial basement membrane components and carotid stiffness in mice.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.

Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.

Vimentin knockout results in increased expression of sub-endothelial basement membrane components and carotid stiffness in mice.

Intermediate filaments are involved in stress-related cell mechanical properties and in plasticity via the regulation of focal adhesions (FAs) and the actomyosin network. We investigated whether vimentin regulates endothelial cells (ECs) and vascular smooth muscle cells (SMCs) and thereby influences vasomotor tone and arterial stiffness. Vimentin knockout mice (Vim-/-) exhibited increased expression of laminin, fibronectin, perlecan, collagen IV and VE-cadherin as well as von Willebrand factor deposition in the subendothelial basement membrane. Smooth muscle (SM) myosin heavy chain, α-SM actin and smoothelin were decreased in Vim-/- mice. Electron microscopy revealed a denser endothelial basement membrane and increased SM cell-matrix interactions. Integrin αv, talin and vinculin present in FAs were increased in Vim-/- mice. Phosphorylated FA kinase and its targets Src and ERK1/2 were elevated in Vim-/- mice. Knockout of vimentin, but not of synemin, resulted in increased carotid stiffness and contractility and endothelial dysfunction, independently of blood pressure and the collagen/elastin ratio. The increase in arterial stiffness in Vim-/- mice likely involves vasomotor tone and endothelial basement membrane organization changes. At the tissue level, the results show the implication of FAs both in ECs and vascular SMCs in the role of vimentin in arterial stiffening.

Temporally controlled onset of dilated cardiomyopathy through disruption of the SRF gene in adult heart.

BACKGROUND: Serum response factor (SRF) is a cardiac transcription factor involved in cell growth and differentiation. We have shown, using the Cre/loxP system, that cardiac-specific disruption of SRF gene in the embryonic heart results in lethal cardiac defects. The role of SRF in adult heart is unknown. METHODS AND RESULTS: We disrupted SRF in the adult heart using a heart-specific tamoxifen-inducible Cre recombinase. This disruption led to impaired left ventricular function with reduced contractility, subsequently progressing to dilated cardiomyopathy, as demonstrated by serial echocardiography, including tissue Doppler imaging. The cytoarchitecture of cardiomyocytes was altered in the intercalated disks. All mutant mice died from heart failure 10 weeks after treatment. These functional and structural defects were preceded by early alterations in the cardiac gene expression program: major decreases in mRNA levels for cardiac alpha-actin, muscle creatine kinase, and calcium-handling genes. CONCLUSIONS: SRF is crucial for adult cardiac function and integrity. We suggest that the rapid progression to heart failure in SRF mutant mice results primarily from decreased expression of proteins involved in force generation and transmission, low levels of polymerized actin, and changes in cytoarchitecture, without hypertrophic compensation. These cardiac-specific SRF-deficient mice have the morphological and clinical features of acquired dilated cardiomyopathy in humans and may therefore be used as an inducible model of this disorder.

Serum Response Factor in Muscle Tissues: From Development to Ageing.

Skeletal, cardiac and smooth muscle cells share various common characteristic features. During development the embryonic mesodermal layer contribute at different proportions to the formation of these tissues. At the functional level, contractility as well as its decline during ageing, are also common features. Cytoskeletal components of these tissues are characterized by various actin isoforms that govern through their status (polymerised versus monomeric) and their interaction with the myosins the contractile properties of these muscles. Finally, at the molecular level, a set of different transcription factors with the notable exception of Serum Response Factor SRF- which is commonly enriched in the 3 types of muscle- drive and maintain the differentiation of these cells (Myf5, MyoD, Myogenin for skeletal muscle; Nkx2.5, GATA4 for cardiomyocytes). In this review, we will focus on the transcription factor SRF and its role in the homeostasis of cardiac, smooth and skeletal muscle tissues as well as its behaviour during the age related remodelling process of these tissues with a specific emphasis on animal models and human data when available.

Mechanism of binding of serum response factor to serum response element.

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124-245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core-SRF labeled with bis-ANS showed that the protein is monomeric at low concentration (10(-7) m). The titration curves for the fluorescence anisotropy of fluorescein-labeled oligonucleotide revealed that under equilibrium conditions, the core-SRF monomers were bound sequentially to SRE at very low concentration (10(-9) m). Curve-fitting data showed also major differences between the wild-type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core-SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core-SRF was bound as a dimer to the wild-type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far-UV CD spectra indicated that the flexibility of core-SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein-labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA-protein interaction.