Cytoskeletal Actin Gene Research Articles

IL-2 delivery to CD8+ T cells during infection requires MRTF/SRF-dependent gene expression and cytoskeletal dynamics

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.

MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation

Actin cytoskeleton is well-known for providing structural/mechanical support, but whether and how it regulates chromatin and cell fate reprogramming is far less clear. Here, we report that MKL1, the key transcriptional co-activator of many actin cytoskeletal genes, regulates genomic accessibility and cell fate reprogramming. The MKL1-actin pathway weakens during somatic cell reprogramming by pluripotency transcription factors. Cells that reprogram efficiently display low endogenous MKL1 and inhibition of actin polymerization promotes mature pluripotency activation. Sustained MKL1 expression at a level seen in typical fibroblasts yields excessive actin cytoskeleton, decreases nuclear volume and reduces global chromatin accessibility, stalling cells on their trajectory toward mature pluripotency. In addition, the MKL1-actin imposed block of pluripotency can be bypassed, at least partially, when the Sun2-containing linker of the nucleoskeleton and cytoskeleton (LINC) complex is inhibited. Thus, we unveil a previously unappreciated aspect of control on chromatin and cell fate reprogramming exerted by the MKL1-actin pathway.

Genomic Instability in Human Pluripotent Stem Cells Arises from Replicative Stress and Chromosome Condensation Defects

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells.

Proteomic analysis of SRF associated transcription complexes identified TFII-I as modulator of SRF function in neurons

The serum response factor (SRF) is a neuronal activity regulated transcription factor (TF) mediating an immediate early (IEGs, e.g. cFos, Egr1) and actin cytoskeletal gene response. SRF activity is adjusted by two competing cofactor families, ternary complex factors (TCF; e.g. Elk-1) and myocardin related transcription factors (MRTF).We investigated mechanisms of SRF activation upon seizure associated neuronal activity in mice. SRF serine 103 phosphorylation or promoter binding was not obviously changed upon neuronal activation. In contrast, a SRF directed proteomic analysis uncovered established and potentially novel components of SRF associated protein complexes whose abundance was modified by neuronal activity. This included the general transcription factor TFII-I for which we provide a first functional analysis in neurons. TFII-I modulated neuronal SRF target gene expression, enhanced nerve fiber growth and modulated the shape of neuronal growth cones. Interestingly, TFII-I modulated two SRF cofactors, Elk-1 and MRTF-A, in opposite directions. TFII-I and Elk-1 co-expression enhanced SRF target gene abundance, SRF promoter binding and neurite growth, whereas TFII-I and MRTF-A resulted in opposite outcomes.In summary, we provide a first proteome wide analysis of SRF associated proteins. We characterized TFII-I as modulator of SRF activity by imposing differential impact on two SRF cofactors.

CNS axon regeneration inhibitors stimulate an immediate early gene response via MAP kinase-SRF signaling.

BackgroundCNS axon regeneration inhibitors such as Nogo and CSPGs (Chondroitin Sulfate Proteoglycans) are major extrinsic factors limiting outgrowth of severed nerve fibers. However, knowledge on intracellular signaling cascades and gene expression programs activated by these inhibitors in neurons is sparse. Herein we studied intracellular signaling cascades activated by total myelin, Nogo and CSPGs in primary mouse CNS neurons.ResultsTotal myelin, Nogo and CSPGs stimulated gene expression activity of the serum response factor (SRF), a central gene regulator of immediate early (IEG) and actin cytoskeletal gene transcription. As demonstrated by pharmacological interference, SRF-mediated IEG activation by myelin, Nogo or CSPGs depended on MAP kinase, to a lesser extent on Rho-GTPase but not on PKA signaling. Stimulation of neurons with all three axon growth inhibitors activated the MAP kinase ERK. In addition to ERK activation, myelin activated the IEG c-Fos, an important checkpoint of neuronal survival vs. apoptosis. Employing Srf deficient neurons revealed that myelin-induced IEG activation requires SRF. This suggests an SRF function in mediating neuronal signaling evoked by axon regeneration associated inhibitors. Besides being a signaling target of axon growth inhibitors, we show that constitutively-active SRF-VP16 can be employed to circumvent neurite growth inhibition imposed by myelin, Nogo and CSPGs.ConclusionIn sum, our data demonstrate that axon regeneration inhibitors such as Nogo trigger gene expression programs including an SRF-dependent IEG response via MAP kinases and Rho-GTPases.

G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.

Serum response factor is an essential transcription factor in megakaryocytic maturation

AbstractSerum response factor (Srf) is a MADS–box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. To test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F knockout (KO) mice are born with normal Mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased number and percentage of CD41+ megakaryocytes (WT: 0.41% ± 0.06%; KO: 1.92% ± 0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation, and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are down-regulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production partly because of regulation of cytoskeletal genes.

Defining a Core Set of Actin Cytoskeletal Proteins Critical for Actin-Based Motility of Rickettsia

Many Rickettsia species are intracellular bacterial pathogens that use actin-based motility for spread during infection. However, while other bacteria assemble actin tails consisting of branched networks, Rickettsia assemble long parallel actin bundles, suggesting the use of a distinct mechanism for exploiting actin. To identify the underlying mechanisms and host factors involved in Rickettsia parkeri actin-based motility, we performed an RNAi screen targeting 115 actin cytoskeletal genes in Drosophila cells. The screen delineated a set of four core proteins-profilin, fimbrin/T-plastin, capping protein, and cofilin--as crucial for determining actin tail length, organizing filament architecture, and enabling motility. In mammalian cells, these proteins were localized throughout R. parkeri tails, consistent with a role in motility. Profilin and fimbrin/T-plastin were critical for the motility of R. parkeri but not Listeria monocytogenes. Our results highlight key distinctions between the evolutionary strategies and molecular mechanisms employed by bacterial pathogens to assemble and organize actin.

Molecular Characterization of Rockbream (Oplegnathus fasciatus) Cytoskeletal β-actin Gene and Its 5'-Upstream Regulatory Region

The cytoskeletal <TEX>$\beta$</TEX>-actin gene and its 5'-upstream region were isolated and characterized in the rockbream (Oplegnathus fasciatus). Complementary DNA of the rockbream <TEX>$\beta$</TEX>-actin represented a 1,125 bp of an open reading frame encoding 375 amino acids, and the rockbream <TEX>$\beta$</TEX>-actin cDNA and deduced amino acid sequences were highly homologous to those of other vertebrate orthologs. At the genomic level, the <TEX>$\beta$</TEX>-actin gene also exhibited an organization typical of vertebrate cytoskeletal actin genes (2,159 bp composed of five translated exons interrupted by four introns) with a conserved GT/AG exon-intron splicing rule. The putative non-translated exon predicted in the rockbream <TEX>$\beta$</TEX>-actin gene was much more homologous with those of teleostean <TEX>$\beta$</TEX>-actin genes than those of mammals. The 5'-upstream regulatory region isolated by genome walking displayed conserved and essential elements such as TATA, CArG and CAAT boxes in its proximal part, while several other immune- or stress-related motifs such as those for NF-kappa B, USF, HNF, AP-1 and C/EBP were in the distal part. Semi-quantitative RT-PCR assay results demonstrated that the rockbream <TEX>$\beta$</TEX>-actin transcripts were ubiquitously but different-tially expressed across the tissues of juveniles.

Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.

Evolution of the ascidian anural larva: evidence from embryos and molecules.

Most ascidians pass through a tadpole (urodele) larval stage, although some species have derived a tailless (anural) larva. New insights into the evolution of anural larvae in the Roscovita clade of molgulid ascidians were obtained from studing embryonic development of the transitional anural species Molgula bleizi and from phylogenetic analysis based on muscle and cytoskeletal actin gene sequences. By observing in vitro fertilized eggs, we found that M. bleizi, previously described as a typical anural developer, actually forms a short immotile tail during embryogenesis. The short tail contains notochord lineage cells, which undergo abbreviated morphogenetic movements but eventually arrest in development. Molgula bleizi tail muscle lineage cells produce the muscle enzyme acetylcholinesterase (AChE) but do not express muscle actin genes. The presence of a short tail, a vestigial notochord, and AChE-positive muscle cells suggest that M. bleizi is a recently derived anural species. An M. bleizi larval muscle actin gene (MbMA1) was isolated, sequenced, and shown to be a pseudogene based on critical deletions in its coding region that would result in a nonfunctional actin protein. The mutations in MbMA1 are distinct from and have evolved independent of the larval muscle actin pseudogenes MoccMA1a and MoccMA1b in Molgula occulta, another anural developer in the Roscovita clade. Pseudogene formation explains the absence of muscle actin mRNA in M. bleizi embryos. The 3' untranslated region of an M. bleizi cytoskeletal actin gene was also isolated and sequenced. Phylogenetic trees reconstructed using muscle and cytoskeletal actin sequences suggest that the anural developer M. bleizi evolved prior to the divergence of the urodele developer Molgula oculata and the anural developer M. occulta in the Roscovita clade. Since M. bleizi lives attached to hard substrata in the tidal zone, whereas M. oculata and M. occulta live buried in subtidal sand flats, our results suggest that the anural larva evolved at least twice in the Roscovita clade of molgulid ascidians as an adaptation to different habitats.

Cytoskeletal actin genes function downstream of HNF-3beta in ascidian notochord development.

We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.

Cis-regulation downstream of cell type specification: a single compact element controls the complex expression of the CyIIa gene in sea urchin embryos.

CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogenic and secondary mesenchyme and, later in development, is permanently activated in the hindgut and midgut. CyIIa transcription follows, and is therefore downstream of, the initial specification of these embryonic domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during development was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regulatory function. Distal elements scattered over several kb account for 60% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron. However, the complex spatial pattern of CyIIa expression is completely reproduced by a compact upstream regulatory element <450 bp in length. We found no evidence anywhere in the 4.4 kb sequence examined for negative regulators required to repress ectopic expression. The specific site that mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types when introduced into embryos of Lytechinus pictus, which belongs to a different echinoid order. No ectopic expression was observed, in contrast to the result of a similar interspecific gene transfer experiment carried out earlier on a different cytoskeletal actin gene that is expressed much earlier in development. Presentation of the set of transcription factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types.

Turnover of histone acetyl groups during sea urchin early development is not required for histone, heat shock and actin gene transcription

Histone acetylation is an extremely complex, reversible and specific process. In order to evaluate the importance of this modification for gene expression during sea urchin development, acetyl group turnover of histone lysine residues was blocked by sodium butyrate. The continuous presence of 15 Mm sodium butyrate in the incubation medium from the onset of development blocked gastrulation and resulted in chromatin containing hyperacetylated histone molecules in amounts usually not found in nature. At the mesenchyme blastula stage, the expression of the early histone genes was shut off and the expression of the late genes was switched on both in control and sodium butyrate-treated embryos. Investigation of the early histone gene chromatin structure in butyrate-treated embryos revealed a random distribution of nucleosomes when the genes were transcriptionally active as compared to regular nucleosomal packaging when genes were inactive. These changes in chromatin structure during development mimicked the chromatin structural transition of the early histone genes in control embryos. In addition, the ability of heat shock genes to be induced at elevated temperature and repressed at normal temperature was unaffected in butyrate treatment of embryos. Finally, the developmental profiles of the cytoskeletal CyIIIa actin gene expression in control and butyrate-treated embryos were very similar. The data presented suggest that turnover of histone acetyl groups and the overall level of histone acetylation are not determining factors in the up and down regulation of a number of genes during early development of sea urchin.

Age-related changes in the mRNA expression of actin isoforms in Drosophila melanogaster.

In all organisms the process of aging is characterized by a gradual loss of homeostatic maintenance functions and physiological fitness. This might be caused-in part-by a down-regulation of important housekeeping and maintenance genes. In our previous work we have shown that in Drosophila melanogaster aging is accompanied by a decrease in steady-state levels of most RNA species. This includes structural genes and housekeeping genes, though at a different level. Here we have examined age-related changes in the mRNA levels of the gene family encoding the six Drosophila actin genes. Two actin isoforms code for cytoskeletal actins and represent housekeeping genes, whereas the four muscle actin genes are expressed tissue-specifically in differentiated muscles. The results suggest that the six actin genes are differentially regulated during aging as well. The two cytoskeletal actin genes show little changes in their steady-state mRNA levels. Drastic changes are observed, however, in the mRNAs coding for muscle actins, particularly the mRNAs coding for the jump and flight muscle actins.

Predominant expression of a cytoskeletal actin gene in mesenchyme cells during embryogenesis of the ascidian Halocynthia roretzi.

During ascidian embryogenesis mesenchymal cells proliferate and have no known function until after metamorphosis, when they give rise to various mesodermal tissues of the adult. Despite their simple lineage, specification mechanisms of the embryonic mesenchyme cells remain to be investigated; this is mainly due to lack of specific molecular markers for this cell type. Here we report that, in Halocynthia roretzi, zygotic expression of a cytoskeletal actin gene (HrCA1) begins at the late gastrula stage and that transcripts were predominantly distributed in embryonic mesenchyme cells, although some expression was observed in special cells of the central nervous system as well as in notochord cells. When HrCA1 expression was examined in cleavage-arrested embryos, it was found only in mesenchyme-lineage blastomeres of arrested 8-cell and later stages, consistent with the predominant expression of HrCA1 being in mesenchyme cells. To examine whether cell-cell contact until the 8-cell stage is required for mesenchyme cell specification, blastomeres were continuously dissociated during the period of 2-8-cell stages and division of the blastomeres was then arrested. Results showed that two of eight dissociated and division-arrested blastomeres from a single fertilized egg (presumably those corresponding to B4.1 cells) expressed HrCA1, suggesting that specification of embryonic mesenchyme cells does not require cell-cell contact until the 8-cell stage.

Spatial and temporal information processing in the sea urchin embryo: modular and intramodular organization of the CyIIIa gene cis-regulatory system

The CyIIIa cytoskeletal actin gene of Strongylocentrotus purpuratus is expressed specifically in the aboral ectoderm. In earlier work we identified a 2.3 kb cis-regulatory region that is necessary and sufficient for correct spatial and temporal expression of a CyIIIa.CAT gene. This region includes about 20 sites of specific protein-DNA interaction, at which at least nine different transcription factors may be bound. All except two of these factors have been cloned. In this work we have analyzed by deletion or mutagenesis each specific interaction. A specific function was identified for every binding site examined. These individual functions include control of amplitude and timing of expression at different phases of embryogenesis, and control of spatial expression. We show that particular negative regulatory interactions are required to repress expression of the CyIIIa.CAT construct in oral ectoderm and in skeletogenic mesenchyme at different stages. In further experiments we determined the overall functional organization of the CyIIIa cis-regulatory system, and we show that this system is modular in its regulatory structure. The 'proximal module' (with respect to the transcription start site) extends upstream for about 800 base pairs, and includes nine target sites serviced by six different transcription factors. Its major role is to establish CyIIIa expression in the aboral ectoderm territory as the blastomere founder cells are specified and the oral-aboral axis is determined, and to activate the CyIIIa gene late in cleavage. The 'middle module,' which lies upstream of the proximal module, acquires major control of CyIIIa function after the blastula stage. It includes six target sites, serviced by four different factors. The middle module is responsible for a sharp increase in expression occurring during gastrulation, mediated by the positively acting factors that bind within it. The middle module also includes sites at which two different negatively acting spatial control factors bind, the functions of which are required for correct spatial expression late in embryogenesis. The 'distal module' contains a number of sites at which a positively acting factor binds, but this module exercises no spatial regulatory function. Interactions within the distal module are required for the normal levels of function of both the proximal and middle modules.

SpZ12-1, a negative regulator required for spatial control of the territory-specific CyIIIa gene in the sea urchin embryo.

The CyIIIa cytoskeletal actin gene of the sea urchin Strongylocentrotus purpuratus is activated in late cleavage and expressed exclusively in the aboral ectoderm territory of the embryo. Previous gene transfer studies defined a 2.3 kb cis-regulatory region that is necessary and sufficient for correct temporal and spatial expression of a CyIIIa.CAT fusion gene. In this paper, a negative regulatory element within this region was identified that is required for repression of the CyIIIa gene in skeletogenic mesenchyme cells. The repression mediated by this regulatory element takes place after initial territorial specification. A cDNA clone encoding a DNA-binding protein with twelve Zn fingers (SpZ12-1) was isolated by probing an expression library with this cis-element. Deletion analysis of the SpZ12-1 protein confirmed that a DNA-binding domain is located within the Zn finger region. SpZ12-1 is the only DNA-binding protein in embryo nuclear extract that interacts with the specific cis-target sites required for repression of CyIIIa.CAT in skeletogenic mesenchyme and is likely to be the trans factor that mediates this repression.

A multimerizing transcription factor of sea urchin embryos capable of looping DNA.

SpGCF1 is a recently cloned sea urchin transcription factor that recognizes target sites in several different sea urchin genes. We find that in gel-shift experiments this factor is able to multimerize. A quantitative simulation of the gel-shift results suggests that SpGCF1 molecules that are bound to DNA target sites may also bind to one another, thus associating several DNA probe molecules. SpGCF1 might therefore be able to loop DNA molecules bearing its target sites at distant locations. We demonstrate this prediction by electron microscopy, and using the well-characterized cis-regulatory domain of the CyIIIa cytoskeletal actin gene, we show that the loop conformations predicted from the known SpGCF1 target site locations are actually formed in vitro. We speculate that the multimerization of this factor in vivo may function to bring distant regions of extended regulatory domains into immediate proximity so that they can interact with one another.

Evolution of actin gene families of sea urchins.

The actin gene family of the sea urchin Lytechinus pictus includes a single muscle actin gene, LpM, and four cytoskeletal actin genes: LpC1, LpC2, LpC3, and LpC4. The origin and relationship of these actin genes to members of the actin gene family of the sea urchin Strongylocentrotus purpuratus were considered. Comparison of deduced amino acid sequences suggested a close relationship between LpC1 and the CyI-CyII subfamily of S. purpuratus actin genes, and between LpC2 and the CyIII subfamily of S. purpuratus actin genes; the muscle actin genes were orthologous. It is proposed that two divergent cytoskeletal actin genes of the common ancestral sea urchin gave rise by duplication to the extant cytoskeletal actin genes of these species, some of which have changed 3' noncoding sequences while others have maintained a terminus highly conserved among sea urchin actin genes.