Defining the mammalian CArGome

Abstract

Serum response factor (SRF) binds a 1216-fold degenerate cis element known as the CArG box. CArG boxes are found primarily in muscle- and growth-factor-associated genes although the full spectrum of functional CArG elements in the genome (the CArGome) has yet to be defined. Here we describe a genome-wide screen to further define the functional mammalian CArGome. A computational approach involving comparative genomic analyses of human and mouse orthologous genes uncovered >100 hypothetical SRF-dependent genes, including 10 previously identified SRF targets, harboring a conserved CArG element within 4000 bp of the annotated transcription start site (TSS). We PCR-cloned 89 hypothetical SRF targets and subjected each of them to at least two of several validations including luciferase reporter, gel shift, chromatin immunoprecipitation, and mRNA expression following RNAi knockdown of SRF; 60/89 (67%) of the targets were validated. Interestingly, 26 of the validated SRF target genes encode for cytoskeletal/contractile or adhesion proteins. RNAi knockdown of SRF diminishes expression of several SRF-dependent cytoskeletal genes and elicits an attending perturbation in the cytoarchitecture of both human and rodent cells. These data illustrate the power of integrating existing algorithms to interrogate the genome in a relatively unbiased fashion for cis-regulatory element discovery. In this manner, we have further expanded the mammalian CArGome with the discovery of an array of cyto-contractile genes that coordinate normal cytoskeletal homeostasis. We suggest one function of SRF is that of an ancient master regulator of the actin cytoskeleton.

Role of serum response factor in the pathogenesis of disease

“…93 Interestingly, a CArG box in the human T-cell leukemia virus-1 that deviates from the conventional sequences, shown in Figure 1, was shown to bind to and be activated by SRF. 94 This would suggest that the full complement of SRF-binding sites in the genome (ie, CArGome 22 ) may be more diverse than previously thought. Indeed, several putative SRF-binding 'CArG boxes' derived from genome-wide binding studies show sequences that diverge substantially from conventional CArG boxes.…”

“…Cells of the dentate gyrus showed a reduction in F-actin, and biochemical data indicated decreases in gelsolin and altered phosphorylation of cofilin, two regulators of cytoskeletal dynamics. While both cofilin genes are direct targets of SRF, 22 the nature of SRF-dependent gelsolin expression is incompletely understood. SRF deletion in the perinatal hippocampus resulted in poor organization and aberrant synapses of the so-called mossy fibers, apparently because of their inability to respond appropriately to guidance cues that drive proper neuronal cell migration and organization.…”

“…[19][20][21][22][23][24][25][26] Bioinformatic and wet-lab assays have disclosed a disproportionate number of SRFtarget genes encoding for elements of the actin cytoskeleton. 20,22,23,26 Evidence for the role of SRF in cytoskeletal processes stems from genetic ablation studies using slug, fly, and worm where loss of SRF function leads to defective animal/cellular locomotion (reviewed by Miano et al 27 ). In fact, SRF is essential for life processes in every species where it has been inactivated, including mice where global loss of SRF leads to incomplete gastrulation and embryonic arrest before organogenesis commences.…”

The serum response factor (SRF)/megakaryocytic acute leukemia (MAL) network participates in megakaryocyte development

“…This may rule out a predominant role of Srf or Mal in their transcriptional regulation in Mks, in contrast to other cell types. 7,20 Expression level of Kindlin-3, which is abundantly expressed in Mks and platelets, was also unaffected. Expression levels were normalized to Gapdh and results were expressed relative to the level of each gene in wild-type Mks (set to 1) (n ¼ 3 per genotype).…”

Differential dependence of stretch and shear stress signaling on caveolin-1 in the vascular wall

“…Levels of cofilin phosphorylation following organ culture were similar in stretched WT and KO portal veins and were reduced to Ͻ50% in the absence of stretch in both genotypes. Expression of the cofilin gene is, like SM22 and desmin, regulated by myocardin and serum response factor (25), and the protein level of cofilin is therefore sensitive to mechanical stretch (3). Furthermore, the Rho kinase inhibitor Y-27632 had similar effects on contractile responses in depolarized WT and KO portal veins.…”