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Smyd1/Bop is an evolutionary conserved histone methyltransferase previously shown by conventional knockout to be critical for embryonic heart development. To further explore the mechanism(s) in a cell autonomous context, we conditionally ablated Smyd1 in the first and second heart fields of mice using a knock-in (KI) Nkx2.5-cre driver. Robust deletion of floxed-Smyd1 in cardiomyocytes and the outflow tract (OFT) resulted in embryonic lethality at E9.5, truncation of the OFT and right ventricle, and additional defects consistent with impaired expansion and proliferation of the second heart field (SHF). Using a transgenic (Tg) Nkx2.5-cre driver previously shown to not delete in the SHF and OFT, early embryonic lethality was bypassed and both ventricular chambers were formed; however, reduced cardiomyocyte proliferation and other heart defects resulted in later embryonic death at E11.5-12.5. Proliferative impairment prior to both early and mid-gestational lethality was accompanied by dysregulation of transcripts critical for endoplasmic reticulum (ER) stress. Mid-gestational death was also associated with impairment of oxidative stress defense-a phenotype highly similar to the previously characterized knockout of the Smyd1-interacting transcription factor, skNAC. We describe a potential feedback mechanism in which the stress response factor Tribbles3/TRB3, when directly methylated by Smyd1, acts as a co-repressor of Smyd1-mediated transcription. Our findings suggest that Smyd1 is required for maintaining cardiomyocyte proliferation at minimally two different embryonic heart developmental stages, and its loss leads to linked stress responses that signal ensuing lethality.

ZIC3, an X-linked zinc finger transcription factor, was the first identified gene involved in establishing normal left-right patterning in humans. Mutations in the Zic3 gene in patients cause heterotaxy, which includes congenital heart defects. However, very little is known about how the function of the ZIC3 protein is regulated. Sumoylation is a posttranslational modification process in which a group of small ubiquitin-like modifier (SUMO) proteins is covalently attached to targets via a series of enzymatic reactions. Here, we report for the first time that sumoylation targets human ZIC3 primarily on the consensus lysine residue K248, which is critical for the nuclear retention of ZIC3. Consequently, SUMO modification potentiates the repressive activity of ZIC3 on the promoter of its target gene cardiac α-actin, and the mutation of lysine 248 to arginine (K248R) abolishes its repressive function. We further revealed that ZIC3 variants with mutations found in human patients with congenital anomalies exhibit aberrant sumoylation activity, which at least partially accounts for their cytoplasmic diffusion. Improved sumoylation of human disease-associated ZIC3 variants reestablishes their nuclear occupancy in the presence of SUMO E3 ligase and SUMO-1. Thus, the altered sumoylation status of ZIC3 underpins the developmental abnormalities associated with these ZIC3 mutants. The SUMO targeting consensus sequence in ZIC3 is highly conserved in its paralogs and orthologs, pointing to sumoylation as a general mechanism underlying the functional control of ZIC proteins. This study provides a potential therapeutic strategy to regain the normal subcellular distribution and function of ZIC3 mutants by restoring SUMO conjugation., (© 2013.)

Nkx2.5 is a cardiac specific homeobox gene critical for normal heart development. We previously identified Nkx2.5 as a target of sumoylation, a posttranslational modification implicated in a variety of cellular activities. Sumoylation enhanced Nkx2.5 activity via covalent attachment to the lysine residue 51, the primary SUMO acceptor site. However, how sumoylation regulates the activity of Nkx2.5 in vivo remains unknown. We generated transgenic mice overexpressing sumoylation deficient mutant K51R (conversion of lysine 51 to arginine) specifically in mouse hearts under the control of cardiac α-myosin heavy chain (α-MHC) promoter (K51R-Tg). Expression of the Nkx2.5 mutant transgene in the wild type murine hearts did not result in any overt cardiac phenotype. However, in the presence of Nkx2.5 haploinsufficiency, cardiomyocyte-specific expression of the Nkx2.5 K51R mutant led to congenital heart diseases (CHDs), accompanied with decreased cardiomyocyte proliferation. Also, a number of human CHDs-associated Nkx2.5 mutants exhibited aberrant sumoylation. Our work demonstrates that altered sumoylation status may underlie the development of human CHDs associated with Nkx2.5 mutants.

Drought is a major limiting factor for plant growth and crop productivity. Many Calcineurin B-like interacting protein kinases (CIPKs) play crucial roles in plant adaptation to environmental stresses. It is particularly essential to find the phosphorylation targets of CIPKs and to study the underlying molecular mechanisms. In this study, we demonstrate that CIPK11 acts as a novel component to modulate drought stress in plants. The overexpression of CIPK11 (CIPK11OE) in Arabidopsis resulted in the decreased tolerance of plant to drought stress. When compared to wild type plants, CIPK11OE plants exhibited higher leaf water loss and higher content of reactive oxygen species (ROS) after drought treatment. Additionally, a yeast two hybrid screening assay by using CIPK11 as a bait captures Di19-3, a Cys2/His2-type zinc-finger transcription factor that is involved in drought stress, as a new interactor of CIPK11. Biochemical analysis revealed that CIPK11 interacted with Di19-3 in vivo and it was capable of phosphorylating Di19-3 in vitro. Genetic studies revealed that the function of CIPK11 in regulating drought stress was dependent on Di19-3. The transcripts of stress responsive genes, such as RAB18, RD29A, RD29B, and DREB2A were down-regulated in the CIPK11OE plants. Whereas overexpression of CIPK11 in di19-3 mutant background, expression levels of those marker genes were not significantly altered. Taken together, our results demonstrate that CIPK11 partly mediates the drought stress response by regulating the transcription factor Di19-3. [ABSTRACT FROM AUTHOR]

Transcriptional regulation is essential for any gene expression including microRNA expression. MiR-1-1 and miR-133a-2 are essential microRNAs (miRs) involved in cardiac and skeletal muscle development and diseases. Early studies reveal two regulatory enhancers, an upstream and an intragenic, that direct the miR-1-1 and miR-133a-2 transcripts. In this study, we identify a unique serum response factor (SRF) binding motif within the enhancer through bioinformatic approaches. This motif is evolutionarily conserved and is present in a range of organisms from yeast, flies, to humans. We provide evidence to demonstrate that this regulatory motif is SRF-dependent in vitro by electrophoretic mobility shift assay, luciferase activity assay, and endogenous chromatin immunoprecipitation assay followed by DNA sequence confirmation, and in vivo by transgenic lacZ reporter mouse studies. Importantly, our transgenic mice indicate that this motif is indispensable for the expression of miR1-1/133a-2 in the heart, but not necessary in skeletal muscle, while the enhancer is sufficient for miR1-1/133a-2 gene expression in both tissues. The mutation of the motif alone completely abolishes miR-1-1/133a-2 gene expression in the animal heart, but not in the skeletal muscle. Our findings reveal an additional architecture of regulatory complex directing miR-1-1/133a-1 gene expression, and demonstrate how this intragenic enhancer differentially manages the expression of the two miRs in the heart and skeletal muscle, respectively. [ABSTRACT FROM AUTHOR]