Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR + . However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca 2+ transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.cardiogenesis | fibroblast reprograming | protein transduction | kinetic imaging

Our conditional serum response factor (SRF) knockout, Srf Cko , in the heart-forming region blocked the appearance of rhythmic beating myocytes, one of the earliest cardiac defects caused by the ablation of a cardiac-enriched transcription factor. The appearance of Hand1 and Smyd1, transcription and chromatin remodeling factors; Acta1, Acta2, Myl3, and Myom1, myofibril proteins; and calcium-activated potassium-channel gene activity (KCNMB1), the channel protein, were powerfully attenuated in the Srf CKO mutant hearts. A requisite role for combinatorial cofactor interactions with SRF, as a major determinant for regulating the appearance of organized sarcomeres, was shown by viral rescue of SRF-null ES cells with SRF point mutants that block cofactor interactions. In the absence of SRF genes associated with biomineralization, GATA-6, bone morphogenetic protein 4 (BMP4), and periostin were strongly up-regulated, coinciding with the down regulation of many SRF dependent microRNA, including miR1, which exerted robust silencer activity over the induction of GATA-6 leading to the down regulation of BMP4 and periostin.heart development ͉ microRNA ͉ periostin ͉ cardiogenesis ͉ GATA6

Herein, the restricted expression of serum response factors (SRF) closely overlapped with Nkx2-5 and GATA4 transcripts in early chick embryos coinciding with the earliest appearance of cardiac ␣-actin (␣CA) transcripts and nascent myocardial cells. The combinatorial expression of SRF, a MADS box factor Nkx2-5 (a NK4 homeodomain), and/or GATA4, a dual C4 zinc finger protein, in heterologous CV1 fibroblasts and Schneider 2 insect cells demonstrated synergistic induction of ␣CA promoter activity. These three factors induced endogenous ␣CA mRNA over a 100-fold in murine embryonic stem cells. In addition, the DNA-binding defective mutant Nkx2-5pm efficiently coactivated the ␣CA promoter in the presence of SRF and GATA4 in the presence of all four SREs and was substantially weakened when individual SREs were mutated and or serially deleted. In contrast, the introduction of SRFpm, a SRF DNA-binding mutant, blocked the activation with all of the ␣CA promoter constructions. These assays indicated a dependence upon cooperative SRF binding for facilitating the recruitment of Nkx2-5 and GATA4 to the ␣CA promoter. Furthermore, the recruitment of Nkx2-5 and GATA4 by SRF was observed to strongly enhance SRF DNA binding affinity. This mechanism allowed for the formation of higher ordered ␣CA promoter DNA binding complexes, led to a model of SRF physical association with Nkx2-5 and GATA4.

The cardiac specific homeobox gene nkx2.5, a member of the nk-2 class family, plays a central role in cardiogenesis and is a target of the small ubiquitin-like modifier (SUMO). Nkx2.5 was modified by SUMO on its 51st amino acid, a lysine residue conserved across species but absent in other nk-2 members. Conversion of this lysine to an arginine (K51R) substantially reduced Nkx2.5 DNA binding and also its transcriptional activity. Unexpectedly, mutant K51R was targeted by ubiquitin. E3 ligase PIAS proteins PIAS1, PIASx, and PIASy, but not PIAS3, enhanced SUMO-1 attachment to Nkx2.5 on the primary SUMO acceptor site. SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins. SUMO conjugation stabilized the formation of Nkx2.5-containing complexes that led to robust transcriptional activation. Thus, SUMO modification serves as a positive regulator for Nkx2.5 transcriptional activity.Cardiac specific homeobox gene nkx2.5 (1, 2), a member of the nk-2 class of homeodomain (HD) 2 factors, is required for early heart development and morphogenesis (3, 4). Nkx2.5, recognized as one of those earliest known markers for cardiac progenitors, has important roles in tissue patterning and lineage determination. Homozygous nkx2.5 null mice died between 9.5 and 11.5 days post coitus (d.p.c.), after the initial heart looping was completed (5, 6), supporting the notion that nkx2.5 played a central role for early cardiac development. Nkx2.5 bound the NKE DNA sequence motif, TYAAGTG, with high affinity via its unique homeodomain that contains a tyrosine at the 54th position in the helix (7). In addition, Nkx2.5 also bound homeodomain target sequences TTAATT, allowing for transactivation of ANF and cardiac ␣-actin promoters (8, 9). Nkx2.5 transcriptional activity may be augmented in combination with other co-factors, such as SRF, GATA4, and Tbx5 (9 -12). Also, posttranslational modification, such as phosphorylation on its serine 163 by casein kinase II, elevated Nkx2.5 activity via increased DNA binding (13). However, little is known about Nkx2.5 functional regulation by other posttranslational modifications.Small ubiquitin-like modifiers (SUMOs) are small molecules that can be covalently and reversibly conjugated to the specific lysine(s) mostly localized in SUMO-targeting sequence KXE (where represents a bulky hydrophobic amino acid, and X represents any residue) within its targets (14, 15). Three functional isoforms of SUMO family members have been recognized in vertebrates, of which active SUMO-2 and -3 share high similarity with each other but only ϳ50% cross-homology with SUMO-1. Also, these SUMO isoforms displayed preferences for different targets. For example, RanGap1 was a preferred substrate by SUMO-1 but poorly modified by . Although the activities of many targets were depressed after SUMO conjugation (14), sumoylation also enhanced the transcriptional activity of some substrates (17)(18)(19)(20). Consequently, SUMO modulates a variety of cellular processes under both physiological and pathological s...

Phosphorylation of a cluster of amino acids in the serum response factor (SRF) ''MADS box'' ␣I coil DNA binding domain regulated the transcription of genes associated with proliferation or terminal muscle differentiation. Mimicking phosphorylation of serine-162, a target of protein kinase C-␣, with an aspartic acid substitution (SRF-S162D) completely inhibited SRF-DNA binding and blocked ␣-actin gene transcription even in the presence of potent myogenic cofactors, while preserving c-fos promoter activity because of stabilization of the ternary complex via Elk-1. Introduction of SRF-S162D into SRF null ES cells permitted transcription of the c-fos gene but was unable to rescue expression of myogenic contractile genes. Transition of proliferating C2C12 myoblasts to postfusion myocytes after serum withdrawal was associated with a progressive decline in SRF-S162 phosphorylation and an increase in ␣-actin gene expression. Hence, the phosphorylation status of serine-162 in the ␣I coil may constitute a novel switch that directs target gene expression into proliferation or differentiation programs.PKC ͉ differentiation ͉ ␣-actin ͉ c-fos

No abstract

Serum response factor (SRF) is a phosphoprotein that regulates skeletal and cardiac alpha-actin gene transcription. Myotonic dystrophy protein kinase (DMPK), a muscle- and neuron-restricted kinase, enhanced SRF-mediated promoter activity of the skeletal and cardiac alpha-actin genes in C2C12 myoblasts as well as in nonmyogenic cells. DMPK phosphorylated SRF in vitro in the alphaI coil of the DNA-binding domain in the MADS box, a highly conserved region required for DNA binding, dimerization, and co-activator interaction in COS and CV1 cells. Threonine 159 in the MADS box alphaI coil was a specific phosphorylation target in vitro as well as in vivo of both DMPK and protein kinase C-alpha. Substitution of threonine 159 with the nonphosphorylatable residue alanine markedly diminished activation of the cardiac alpha-actin promoter in the presence of kinase, while its substitution with aspartic acid, to introduce a negative charge and mimic phosphorylation, restored activation completely. Phosphorylation of the MADS box may constitute a novel mechanism for regulation of SRF-dependent actin gene transcription.