Tceal7 Regulates Skeletal Muscle Development through Its Interaction with Cdk1.

We have previously reported Tceal7 as a muscle-specific gene that represses myoblast proliferation and promotes myogenic differentiation. The regulatory mechanism of Tceal7 gene expression has been well clarified recently. However, the underlying mechanism of Tceal7 function in skeletal muscle development remains to be elucidated. In the present study, we have generated an MCK 6.5 kb-HA-Tceal7 transgenic model. The transgenic mice are born normally, while they have displayed defects in the growth of body weight and skeletal muscle myofiber during postnatal development. Although four RxL motifs have been identified in the Tceal7 protein sequence, we have not detected any direct protein-protein interaction between Tceal7 and Cyclin A2, Cyclin B1, Cylin D1, or Cyclin E1. Further analysis has revealed the interaction between Tceal7 and Cdk1 instead of Cdk2, Cdk4, or Cdk6. Transgenic overexpression of Tceal7 reduces phosphorylation of 4E-BP1 Ser65, p70S6K1 Thr389, and Cdk substrates in skeletal muscle. In summary, these studies have revealed a novel mechanism of Tceal7 in skeletal muscle development.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

AIMS: Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. METHODS AND RESULTS: We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/ß-catenin signalling pathway and thereby promoting differentiation. CONCLUSION: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

Transcription Regulation of Tceal7 by the Triple Complex of Mef2c, Creb1 and Myod.

Tceal7 has been identified as a direct, downstream target gene of MRF in the skeletal muscle. The overexpression of Tceal7 represses myogenic proliferation and promotes cell differentiation. Previous studies have defined the 0.7 kb upstream fragment of the Tceal7 gene. In the present study, we have further determined two clusters of transcription factor-binding motifs in the 0.7 kb promoter: CRE#2-E#1-CRE#1 in the proximal region and Mef2#3-CRE#3-E#4 in the distal region. Utilizing transcription assays, we have also shown that the reporter containing the Mef2#3-CRE#3-E#4 motifs is synergistically transactivated by Mef2c and Creb1. Further studies have mapped out the protein-protein interaction between Mef2c and Creb1. In summary, our present studies support the notion that the triple complex of Mef2c, Creb1 and Myod interacts with the Mef2#3-CRE#3-E#4 motifs in the distal region of the Tceal7 promoter, thereby driving Tceal7 expression during skeletal muscle development and regeneration.

AFM Imaging Reveals Multiple Conformational States of ADAMTS13.

BACKGROUND: ADAMTS13 (A disintegrin and metalloprotease with a thrombospondin type 1 motif 13) cleaves Von Willebrand factor (VWF) to regulate its size, thereby preventing aberrant platelet aggregation and thrombus. Deficiency of ADAMTS13 caused by either genetic mutations or by inhibitory autoantibodies against ADAMTS13 leads to thrombotic thrombocytopenic purpura (TTP). Recently, ADAMTS13 was reported to adopt a "closed" conformation with lower activity and an "open" one resulting from the engagements of VWF D4-CK domains or antibodies to the distal domains of ADAMTS13, or mutations in its spacer domain. These engagements or mutations increase ADAMTS13 activity by ~ 2.5-fold. However, it is less known whether the conformation of ADAMTS13 is dynamic or stable. RESULTS: Wild type ADAMTS13 (WT-ADAMTS13) and the gain-of-function variant (GOF-ADAMTS13) with five mutations (R568K / F592Y / R660K / Y661F / Y665F) in spacer domain were imaged by atomic force microscopy (AFM) at pH 6 and pH 7.5. The data revealed that at both pH 6 and pH 7.5, WT-ADAMTS13 adopted two distinct conformational states (state I and state II), while an additional state (state III) was observed in GOF-ADAMTS13. In the present study, we propose that state I is the "closed" conformation, state III is the "open" one, and state II is an intermediate one. Comparing to pH 7.5, the percentages of state II of WT-ADAMTS13 and state III of GOF-ADAMTS13 increased at pH 6, with the decrease in the state I for WT-ADAMTS13 and state I and state II for GOF-ADAMTS13, suggesting lower pH extended the conformation of ADAMTS13. CONCLUSION: Both WT- and GOF-ADAMTS13 exist multiple conformational states and lower pH might alter the tertiary structure and/or disrupt the intra-domain interactions, increasing the flexibility of ADAMTS13 molecules.

The Etv2-miR-130a Network Regulates Mesodermal Specification.

MicroRNAs (miRNAs) are known to regulate critical developmental stages during embryogenesis. Here, we defined an Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors toward the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without affecting hematopoiesis. Mechanistically, miR-130a directly suppressed Pdgfra expression and promoted the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a overexpression and knockout phenotypes, respectively. In summary, we report the function of a miRNA that specifically promotes the divergence of the hemato-endothelial progenitor to the endothelial lineage.

Feedback Mechanisms Regulate Ets Variant 2 (Etv2) Gene Expression and Hematoendothelial Lineages.

Etv2 is an essential transcriptional regulator of hematoendothelial lineages during embryogenesis. Although Etv2 downstream targets have been identified, little is known regarding the upstream transcriptional regulation of Etv2 gene expression. In this study, we established a novel methodology that utilizes the differentiating ES cell and embryoid body system to define the modules and enhancers embedded within the Etv2 promoter. Using this system, we defined an autoactivating role for Etv2 that is mediated by two adjacent Ets motifs in the proximal promoter. In addition, we defined the role of VEGF/Flk1-Calcineurin-NFAT signaling cascade in the transcriptional regulation of Etv2. Furthermore, we defined an Etv2-Flt1-Flk1 cascade that serves as a negative feedback mechanism to regulate Etv2 gene expression. To complement and extend these studies, we demonstrated that the Flt1 null embryonic phenotype was partially rescued in the Etv2 conditional knockout background. In summary, these studies define upstream and downstream networks that serve as a transcriptional rheostat to regulate Etv2 gene expression.

Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex.

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.

The transcription factor Mesp1 interacts with cAMP-responsive element binding protein 1 (Creb1) and coactivates Ets variant 2 (Etv2) gene expression.

Mesoderm posterior 1 (Mesp1) is well recognized for its role in cardiac development, although it is expressed broadly in mesodermal lineages. We have previously demonstrated important roles for Mesp1 and Ets variant 2 (Etv2) during lineage specification, but their relationship has not been defined. This study reveals that Mesp1 binds to the proximal promoter and transactivates Etv2 gene expression via the CRE motif. We also demonstrate the protein-protein interaction between Mesp1 and cAMP-responsive element binding protein 1 (Creb1) in vitro and in vivo. Utilizing transgenesis, lineage tracing, flow cytometry, and immunostaining technologies, we define the lineage relationship between Mesp1- and Etv2-expressing cell populations. We observe that the majority of Etv2-EYFP(+) cells are derived from Mesp1-Cre(+) cells in both the embryo and yolk sac. Furthermore, we observe that the conditional deletion of Etv2, using a Mesp1-Cre transgenic strategy, results in vascular and hematopoietic defects similar to those observed in the global deletion of Etv2 and that it has embryonic lethality by embryonic day 9.5. In summary, our study supports the hypothesis that Mesp1 is a direct upstream transactivator of Etv2 during embryogenesis and that Creb1 is an important cofactor of Mesp1 in the transcriptional regulation of Etv2 gene expression.

Sox7 is regulated by ETV2 during cardiovascular development.

Vasculogenesis/angiogenesis is one of the earliest processes that occurs during embryogenesis. ETV2 and SOX7 were previously shown to play a role in endothelial development; however, their mechanistic interaction has not been defined. In the present study, concomitant expression of Etv2 and Sox7 in endothelial progenitor cells was verified. ETV2 was shown to be a direct upstream regulator of Sox7 that binds to ETV2 binding elements in the Sox7 upstream regulatory region and activates transcription. We observed that SOX7 over-expression can mimic ETV2 and increase endothelial progenitor cells in embryonic bodies (EBs), while knockdown of Sox7 is able to block ETV2-induced increase in endothelial progenitor cell formation. Angiogenic sprouting was increased by ETV2 over-expression in EBs, and it was significantly decreased in the presence of Sox7 shRNA. Collectively, these studies support the conclusion that ETV2 directly regulates Sox7, and that ETV2 governs endothelial development by regulating transcriptional networks which include Sox7.

Cooperative interaction of Etv2 and Gata2 regulates the development of endothelial and hematopoietic lineages.

Regulatory mechanisms that govern lineage specification of the mesodermal progenitors to become endothelial and hematopoietic cells remain an area of intense interest. Both Ets and Gata factors have been shown to have important roles in the transcriptional regulation in endothelial and hematopoietic cells. We previously reported Etv2 as an essential regulator of vasculogenesis and hematopoiesis. In the present study, we demonstrate that Gata2 is co-expressed and interacts with Etv2 in the endothelial and hematopoietic cells in the early stages of embryogenesis. Our studies reveal that Etv2 interacts with Gata2 in vitro and in vivo. The protein-protein interaction between Etv2 and Gata2 is mediated by the Ets and Gata domains. Using the embryoid body differentiation system, we demonstrate that co-expression of Gata2 augments the activity of Etv2 in promoting endothelial and hematopoietic lineage differentiation. We also identify Spi1 as a common downstream target gene of Etv2 and Gata2. We provide evidence that Etv2 and Gata2 bind to the Spi1 promoter in vitro and in vivo. In summary, we propose that Gata2 functions as a cofactor of Etv2 in the transcriptional regulation of mesodermal progenitors during embryogenesis.

Kbtbd5 is regulated by MyoD and restricted to the myogenic lineage.

BTB-BACK-Kelch (BBK) proteins play broad roles in cellular and molecular regulation. The role of BBK proteins in the skeletal muscle lineage and myogenesis remains an active area of research. Herein, we report a novel BBK gene, Kbtbd5, which we discovered and found to be restricted to the myogenic lineage. We observed that Kbtbd5 was absent in proliferating myoblasts and upregulated upon myogenic differentiation. In situ hybridization analysis revealed that Kbtbd5 was restricted to the skeletal muscle lineage during embryogenesis. We identified a conserved 1.2kb upstream region, which directs reporter expression to the developing skeletal muscle lineage. Transcriptional and mutagenesis assays demonstrated that the E-box motifs contribute to the Kbtbd5 promoter activity. We have also demonstrated the in vivo and in vitro binding between MRFs and the E-box motif in the 1.2kb promoter of the Kbtbd5 gene. Our studies have revealed that the Myod family can transactivate the 1.2kb-luc reporter through the E-box motifs. In addition, we have shown that Kbtbd5 can recruit the Cullin 3 complex in vivo. Using shRNA knockdown, our study has revealed that Kbtbd5 plays an important role in the myogenic differentiation. In summary, we have demonstrated that Kbtbd5 is the direct downstream target gene of the Myod family and regulates myogenic differentiation. Our results further support the notion that Kbtbd5 may serve as an adapter of Cul3 during myogenic differentiation.

Mesp1 patterns mesoderm into cardiac, hematopoietic, or skeletal myogenic progenitors in a context-dependent manner.

Mesp1 is regarded as the master regulator of cardiovascular development, initiating the cardiac transcription factor cascade to direct the generation of cardiac mesoderm. To define the early embryonic cell population that responds to Mesp1, we performed pulse inductions of gene expression over tight temporal windows following embryonic stem cell differentiation. Remarkably, instead of promoting cardiac differentiation in the initial wave of mesoderm, Mesp1 binds to the Tal1 (Scl) +40 kb enhancer and generates Flk-1+ precursors expressing Etv2 (ER71) and Tal1 that undergo hematopoietic differentiation. The second wave of mesoderm responds to Mesp1 by differentiating into PDGFRα+ precursors that undergo cardiac differentiation. Furthermore, in the absence of serum-derived factors, Mesp1 promotes skeletal myogenic differentiation. Lineage tracing revealed that the majority of yolk sac and many adult hematopoietic cells derive from Mesp1+ precursors. Thus, Mesp1 is a context-dependent determination factor, integrating the stage of differentiation and the signaling environment to specify different lineage outcomes.

Etv2 rescues Flk1 mutant embryoid bodies.

Independent mouse knockouts of Etv2 and Flk1 are embryonic lethal and lack hematopoietic and endothelial lineages. We previously reported that Flk1 activates Etv2 in the initiation of hematopoiesis and vasculogenesis. However, Flk1 and its ligand VEGF are expressed throughout development, from E7.0 to adulthood, whereas Etv2 is expressed only transiently during embryogenesis. These observations suggest a complex regulatory interaction between Flk1 and Etv2. To further examine the Flk1 and Etv2 regulatory interaction, we transduced Etv2 and Flk1 mutant ES cells with viral integrants that inducibly overexpress Flk1 or Etv2. We demonstrated that forced expression of Etv2 rescued the hematopoietic and endothelial potential of differentiating Flk1 and Etv2 mutant cells. We further discovered that forced expression of Flk1 can rescue that of the Flk1, but not Etv2 mutant cells. Therefore, we conclude that the requirement for Flk1 can be bypassed by expressing Etv2, supporting the notion that disruption of Etv2 expression is responsible for the early phenotypes of the Etv2 and Flk1 mutant embryos.

VEGF/Flk1 signaling cascade transactivates Etv2 gene expression.

Previous reports regarding the genetic hierarchy between Ets related protein 71 (Er71/Etv2) and Flk1 is unclear. In the present study, we pursued a genetic approach to define the molecular cascade between Etv2 and Flk1. Using a transgenic Etv2-EYFP reporter mouse, we examined the expression pattern of Etv2 relative to Flk1 in the early conceptus. Etv2-EYFP was expressed in subset of Flk1 positive cells during primitive streak stages, suggesting that Flk1 is upstream of Etv2 during gastrulation. Analysis of reporter gene expression in Flk1 and Etv2 mutant mice further supports the hypothesis that Flk1 is necessary for Etv2 expression. The frequency of cells expressing Flk1 in Etv2 mutants is only modestly altered (21% decrease), whereas expression of the Etv2-EYFP transgenic reporter was severely reduced in the Flk1 null background. We further demonstrate using transcriptional assays that, in the presence of Flk1, the Etv2 promoter is activated by VEGF, the Flk1 ligand. Pharmacological inhibition studies demonstrate that VEGF mediated activation is dependent on p38 MAPK, which activates Creb. We identify the VEGF response element in the Etv2 promoter and demonstrate that Creb binds to this motif by EMSA and ChIP assays. In summary, we provide new evidence that VEGF activates Etv2 by signaling through Flk1, which activates Creb through the p38 MAPK signaling cascade.

Foxk1 promotes cell proliferation and represses myogenic differentiation by regulating Foxo4 and Mef2.

In response to severe injury, adult skeletal muscle exhibits a remarkable regenerative capacity due to a resident muscle stem/progenitor cell population. While a number of factors are expressed in the muscle progenitor cell (MPC) population, the molecular networks that govern this cell population remain an area of active investigation. In this study, utilizing knockdown techniques and overexpression of Foxk1 in the myogenic lineage, we observed dysregulation of Foxo and Mef2 downstream targets. Utilizing an array of technologies, we establish that Foxk1 represses the transcriptional activity of Foxo4 and Mef2 and physically interacts with Foxo4 and Mef2, thus promoting MPC proliferation and antagonizing the myogenic lineage differentiation program, respectively. Correspondingly, knockdown of Foxk1 in C2C12 myoblasts results in cell cycle arrest, and Foxk1 overexpression in C2C12CAR myoblasts retards muscle differentiation. Collectively, we have established that Foxk1 promotes MPC proliferation by repressing Foxo4 transcriptional activity and inhibits myogenic differentiation by repressing Mef2 activity. These studies enhance our understanding of the transcriptional networks that regulate the MPC population and muscle regeneration.

Foxk1 recruits the Sds3 complex and represses gene expression in myogenic progenitors.

Previous studies have established that Foxk1 (forkhead box k1) plays an important role in skeletal muscle regeneration. Foxk1 regulates the cell-cycle progression of myogenic progenitors by repressing the cell-cycle inhibitor gene p21. However, the underlying mechanism is not well understood. In the present study, we report the identification of Sds3 (suppressor of defective silencing 3) as an adaptor protein that recruits the Sin3 [SWI (switch)-independent 3]-HDAC (histone deacetylase) repression complex and binds Foxk1. Using GST (glutathione transferase) pull-down assays, we defined the interaction between the Foxk1 FHA (forkhead-associated domain) domain and phospho-Thr(49) in Sds3. We demonstrated that the transcriptional repression of Foxk1 is dependent on the Sin3-Sds3 repression complex, and knockdown of Sds3 results in cell-cycle arrest. We further identified the protein kinase CK2 as the protein kinase for Sds3 Thr(49) and demonstrated that the protein kinase activity of CK2 is required for proper cell-cycle progression. Analysis of CK2 mutant mice reveals perturbation of skeletal muscle regeneration due to the dysregulation of cell-cycle kinetics. Overall, these studies define a CK2-Sds3-Foxk1 cascade that modulates gene expression and regulates skeletal muscle regeneration.

Etv2 is expressed in the yolk sac hematopoietic and endothelial progenitors and regulates Lmo2 gene expression.

During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages; however, the precise roles of Etv2 in yolk sac development remains unclear. In this study, we define the role of Etv2 in yolk sac blood island development using the Etv2 mutant and a novel Etv2-EYFP reporter transgenic line. Both the hematopoietic and the endothelial lineages are absent in the Etv2 mutant yolk sac. In the Etv2-EYFP transgenic mouse, the EYFP reporter is activated in the nascent mesoderm, expressed in the endothelial and blood progenitors, and in the Tie2(+), c-kit(+), and CD41(+) hematopoietic population. The hematopoietic activity in the E7.75 yolk sac was exclusively localized to the Etv2-EYFP(+) population. In the Etv2 mutant yolk sac, Tie2(+) cells are present but do not express hematopoietic or endothelial markers. In addition, these cells do not form hematopoietic colonies, indicating an essential role of Etv2 in the specification of the hematopoietic lineage. Forced overexpression of Etv2 during embryoid body differentiation induces the hematopoietic and the endothelial lineages, and transcriptional profiling in this context identifies Lmo2 as a downstream target. Using electrophoretic mobility shift assay, chromatin immunoprecipitation, transcriptional assays, and mutagenesis, we demonstrate that Etv2 binds to the Lmo2 enhancer and transactivates its expression. Collectively, our studies demonstrate that Etv2 is expressed during and required for yolk sac hematoendothelial development, and that Lmo2 is one of the downstream targets of Etv2.

Sin3 interacts with Foxk1 and regulates myogenic progenitors.

We have previously reported Foxk1 as an important transcription factor in the myogenic progenitors. SWI-independent-3 (Sin3) has been identified as a Foxk1 binding candidate using a yeast two-hybrid screen. In the present study, we have identified the Foxk1 N-terminal (1-40) region as the Sin3 interacting domain (SID), and the PAH2 of Sin3 as the Foxk1 binding domain utilizing yeast two-hybrid and GST pull-down assays. Further studies revealed that knockdown of Sin3a or Sin3b results in cell cycle arrest and upregulation of cell cycle inhibitor genes. In summary, our present studies have shown that Foxk1 interacts with Sin3 through the SID and that Sin3 has an important role in the regulation of cell cycle kinetics of the MPC population. The results of these studies continue to define and assemble the networks that regulate the MPCs and muscle regeneration.

ER71 directs mesodermal fate decisions during embryogenesis.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.