The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardininduced transactivation was not observed in SRF ؊/؊ ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22␣ promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22␣, smooth muscle ␣-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.The diverse functions mediated by smooth muscle cells (SMCs) in organ systems throughout the body are ultimately dependent upon the expression of a unique set of SMC-restricted contractile and cytoskeletal proteins that distinguish this cell lineage from cardiac and skeletal myocytes. A distinguishing feature of the SMC lineage is the capacity of SMCs to reversibly modulate their phenotype and proliferate in response to a variety of stimuli during postnatal development (for a review, see reference 35). In the vasculature, SMCs in the tunica medium of arteries and veins are cell cycle arrested and express a set of lineage-restricted genes, including those for smooth muscle (SM) myosin heavy chain, SM ␣-actin, SM22␣, and calponin, which together define the unique contractile properties of this muscle cell lineage. However, in response to arterial injury, SMCs downregulate expression of contractile genes and concomitantly upregulate a set of genes required for synthetic, migratory, and proliferative functions. This phenotypic modulation has been implicated in the pathogenesis of diseases, including atherosclerosis, restenosis following coronary angioplasty and/or stent implantation, pulmonary hypertension, and asthma (14,33,39,41).Because it is expressed exclusively and abundantly in SMCs during postnatal development (23, 42), our group and others have utilized the SMC-restricted SM22␣ promoter as a model system to elucidate the molecular mechanisms that regulate SMC differentiation and modulation of the SMC phenotype (4,19,24,28,34,42,44). The 441-bp mo...

Unlike human L1 retrotransposons, the 5′ UTR of mouse L1 elements contains tandem repeats of ∼200 bp in length called monomers. Multiple L1 subfamilies exist in the mouse which are distinguished by their monomer sequences. We previously described a young subfamily, called the TF subfamily, which contains ∼1800 active elements among its 3000 full-length members. Here we characterize a novel subfamily of mouse L1 elements, GF, which has unique monomer sequence and unusual patterns of monomer organization. A majority of these GF elements also have a unique length polymorphism in ORF1. Polymorphism analysis of GF elements in various mouse subspecies and laboratory strains revealed that, like TF, the GF subfamily is young and expanding. About 1500 full-length GF elements exist in the diploid mouse genome and, based on the results of a cell culture assay, ∼400 GF elements are potentially capable of retrotransposition. We also tested 14 A-type subfamily elements in the assay and estimate that about 900 active A elements may be present in the mouse genome. Thus, it is now known that there are three large active subfamilies of mouse L1s; TF, A, and GF, and that in total ∼3000 full-length elements are potentially capable of active retrotransposition. This number is in great excess to the number of L1 elements thought to be active in the human genome

The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.

The SAP domain transcription factor myocardin plays a critical role in the transcriptional program regulating smooth muscle cell differentiation. In this report, we describe the capacity of myocardin to physically associate with megakaryoblastic leukemia factor-1 (MKL1) and characterize the function of MKL1 in smooth muscle cells (SMCs). The MKL1 gene is expressed in most human tissues and myocardin and MKL are co-expressed in SMCs. MKL1 and myocardin physically associate via conserved leucine zipper domains. Overexpression of MKL1 transactivates serum response factor (SRF)-dependent SMC-restricted transcriptional regulatory elements including the SM22␣ promoter, smooth muscle myosin heavy chain promoter/enhancer, and SM-␣-actin promoter/enhancer in non-SMCs. Moreover, forced expression of MKL1 and SRF in undifferentiated SRF ؊/؊ embryonic stem cells activates multiple endogenous SMC-restricted genes at levels equivalent to, or exceeding, myocardin. Forced expression of a dominant-negative MKL1 mutant reduces myocardin-induced activation of the SMC-specific SM22␣ promoter. In NIH3T3 fibroblasts MKL1 localizes to the cytoplasm and translocates to the nucleus in response to serum stimulation, actin treadmilling, and RhoA signaling. In contrast, in SMCs MKL1 is observed exclusively in the nucleus regardless of serum conditions or RhoA signaling. However, when actin polymerization is disrupted MKL1 translocates from the nucleus to the cytoplasm in SMCs. Together, these data were consistent with a model wherein MKL1 transduces signals from the cytoskeleton to the nucleus in SMCs and regulates SRF-dependent SMC differentiation autonomously or in concert with myocardin.

SM22␣ is a 22-kDa smooth muscle cell (SMC) lineage-restricted protein that physically associates with cytoskeletal actin filament bundles in contractile SMCs. To examine the function of SM22␣, gene targeting was used to generate SM22␣-deficient (SM22 ؊/؊LacZ ) mice. The gene targeting strategy employed resulted in insertion of the bacterial lacZ reporter gene at the SM22␣ initiation codon, permitting precise analysis of the temporal and spatial pattern of SM22␣ transcriptional activation in the developing mouse. Northern and Western blot analyses confirmed that the gene targeting strategy resulted in a null mutation. Histological analysis of SM22 ؉/؊LacZ embryos revealed detectable ␤-galactosidase activity in the unturned embryonic day 8.0 embryo in the layer of cells surrounding the paired dorsal aortae concomitant with its expression in the primitive heart tube, cephalic mesenchyme, and yolk sac vasculature. Subsequently, during postnatal development, ␤-galactosidase activity was observed exclusively in arterial, venous, and visceral SMCs. SM22␣-deficient mice are viable and fertile. Their blood pressure and heart rate do not differ significantly from their control SM22␣؉/؊ and SM22␣ ؉/؉ littermates. The vasculature and SMC-containing tissues of SM22␣-deficient mice develop normally and appear to be histologically and ultrastructurally similar to those of their control littermates. Taken together, these data demonstrate that SM22␣ is not required for basal homeostatic functions mediated by vascular and visceral SMCs in the developing mouse. These data also suggest that signaling pathways that regulate SMC specification and differentiation from local mesenchyme are activated earlier in the angiogenic program than previously recognized.