Identification of CaMKII-dependent H3S28p target genes
Ontology highlight
ABSTRACT: To identify direct CaMKII-dependent downstream genes of H3S28p we used chromatin-immunoprecipitation followed by massive parallel DNA sequencing (ChIPseq) analysis of isolated adult murine cardiomyocytes from CamKII WT as well as from CaMKIIγ/CaMKIIδ double knockout (DKO) . DKO and WT cells were subjected to long-term Iso treatment for 24 h to maximize CaMKII-dependent H3S28p.
Project description:Histone Deacetylase 4 (HDAC4) is known to contribute to cardiac remodeling processes. We wanted to identify and compare different genome wide targets of HDAC4 to well described interaction partners and its functional consequences. Therefore, we used a model of adult cardiomyocytes isolated from HDAC4 knockout mice and Wildtyp (WT) controls. We looked for HDAC4s contribution to changes of activating histone modifications (H3K4me3, H3K9ac, H3K27ac) and repressive pendants (H3K9me2 and H3K27me3).
Project description:The experiment was designed to assess genome-wide binding sites of Myocyte Enhancer Factor 2 (MEF2) in adult mouse ventricular cardiomyocytes.
Project description:Myocyte enhancer factor 2 contributes as a transcription factor to cardiac remodeling processes. We wanted to link and compare known myocyte enhancer factor 2 targets to overall targets. Therefore, we used a model of neonatal rat cardiomyocytes and precipitated sheared chromatin samples with 5µg of MEF2 Ab.
Project description:We previously found that mice with heterozygous knockout of the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII HKO mice) show various dysregulated behaviors, including cyclic variations in locomotor activity (LA), suggesting that alpha-CaMKII HKO mice may serve as an animal model showing infradian oscillation of mood. We performed gene expression microarray analysis of dentate gyrus from alpha-CaMKII HKO mice. Mice were selected for the sampling such that their LA levels varied among the mice.
Project description:Purpose: The goal of this study was to use deep sequencing to identify all splice variants of Calcium/calmodulin-dependent kinase II (CaMKII) expressed in the human hippocampus. Methods: Transcripts of CaMKII-encoding genes (CAMK2A, CAMK2B, CAMK2G, and CAMK2D) were sub-amplified by PCR from total RNA extracted from human hippocampal tissue samples from 3 donors. Illumina sequencing libraries were constructed by PCR from these initial pools of amplicons and sequenced on an Illumina MiSeq instrument. Sequencing reads passing quality controls were clustered on the basis of sequence identity or near-identity. Consensus sequences of clusters were mapped with known exons of CaMKII genes to identify the splice variant represented by each cluster. Donor 1 replicate 2, Donor 2, and Donor 3 libraries from genes CAMK2B, CAMK2G, and CAMK2D were first sequenced on a MiSeq Nano flow cell, then re-pooled for read balancing and sequenced again of a full-size MiSeq flow cell. For each library, reads from Nano and full-size flow cells were combined for subsequent analysis. Results: We perfomed the first comprehensive survey of CaMKII transcripts expressed in individual tissue samples (human hippocampus). We detected a total of 79 splice variants of the four human CaMKIIs: CaMKIIα (3), CaMKIIβ (30), CaMKIIγ (24), and CaMKIIδ (22), across tissue samples from 3 donors. This represents the vast majority of possible in-frame CaMKII splice variants (Sloutsky and Stratton, European Journal of Neuroscience, 2020; https://doi.org/10.1111/ejn.14761).
Project description:We previously found that mice with heterozygous knockout of the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII HKO mice) show various dysregulated behaviors, including cyclic variations in locomotor activity (LA), suggesting that alpha-CaMKII HKO mice may serve as an animal model showing infradian oscillation of mood. We performed gene expression microarray analysis of dentate gyrus from alpha-CaMKII HKO mice. Mice were selected for the sampling such that their LA levels varied among the mice. Dentate gyrus RNA isolated from alpha-CaMKII HKO mice.
Project description:Mice overexpressing CaMKII in myocardial mitochondria have dilated cardiomyopathy that appears to be exclusively driven by metabolic defects that lead to reduced ATP production. Phosphoproteomics study of mitochondrial proteins supports the metabolomics data.
Project description:Purpose: Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brain stem, and spinal cord, clinical symptoms appear quite variable and a multitude of gene variants have been associated with the disease. Here, Dars2 deletion from CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at 5 months, and culminating by 9 months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Methods: Cortical samples were collected from DARS2-Flox/CamKII-Cre(+) and DARS2-Flox/CamKII-Cre(-) at 14 weeks of age. RNA-seq libraries were prepared using purified RNA isolated from frozen tissue using the RNeasy Mini kit. RNA quality and concentration were assayed using a Fragment Analyzer instrument. RNA-seq libraries were prepared using the TruSeq Stranded mRNA PolyA (Unique Dual Index) kit.. Libraries were sequenced on an Illumina HiSeq 4000 instrument. Samples were taken from N=7 DARS2-Flox/CamKII-Cre(+) and N=6 DARS2-Flox/CamKII-Cre(-) male mice. Conclusions: RNA-seq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and the defense response regulation. Among these transcripts, Cystatin F (Cst7), a protease inhibitor recognized as a marker of neurodegenerative disease and active demyelination, is upregulated over 200-fold in mutant mice and may serve as a key regulator of disease progression.
Project description:The Ca2+/calmodulin-dependent kinase II is expressed in smooth muscle and believed to mediate intracellular calcium handling and calcium-dependent gene transcription. CaMKII is activated by Angiotensin-II. The multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by Angiotensin-II (Ang-II) in vascular smooth muscle cells (VSMC), but its impact on hypertension remains unknown. In our transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), the blood pressure response to chronic Ang-II infusion was significantly reduced as compared to littermate controls. Surprisingly, examination of blood pressure and heart rate under ganglionic blockade revealed a key role for VSMC CaMKII in efferent sympathetic outflow in response to Ang II hypertension. Consistently, the efferent splanchnic nerve activity and plasma phenylephrine concentrations were significantly lower in TG SM-CaMKIIN mice as compared to littermates. Moreover, the aortic depressor nerve activity was reset in hypertensive wild type animals, but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor wall activity may be responsible for the blood pressure difference in Ang-II hypertension. The pulse wave velocity, a measure of vascular wall stiffness in vivo, was increased in aortas of hypertensive compared to normotensive WT animals. However, Ang-II infusion did not alter the pulse wave velocity in transgenic mice, suggesting that CaMKII in VSMC controls structural smooth muscle genes. Accordingly, analysis of gene expression changes in aortas from wild type and TG SM-CaMKIIN hypertensive mice demonstrated that CaMKII inhibition mainly altered the expression of muscle contractile proteins. In contrast, TG SM-CaMKIIN aortas were protected from the Ang-II induced upregulation of genes linked to proliferation, suggesting that CaMKII inhibition prevents the Ang-II-induced reprogramming of smooth muscle cell gene expression towards a proliferative phenotype.
Project description:The scope of the analysis was to discriminate between CaMKII-dependent and independent targets of Wnt3a stimulation in human articular chondrocytes. Chondrocytes were isolated from preserved areas of the articular cartilage removed from 4 patients affected by osteoarthritis. The samples were stimulated with KN92 (inactive analogue of the CaMKII inhibitor KN93), KN92+Wnt3a and KN93+Wnt3a.