Matthias D. Seidl

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

The Cyclic AMP Response Element Modulator α Suppresses CD86 Expression and APC Function

The cAMP response element modulator (CREM)α is a widely expressed transcriptional repressor that is important for the termination of the T cell immune response and contributes to the abnormal T cell function in patients with systemic lupus erythematosus. We present evidence that APCs of Crem−/− mice express increased amounts of the costimulatory molecule CD86 and induce enhanced Ag-dependent and Ag-independent T cell proliferation. Similarly, human APCs in which CREMα was selectively suppressed expressed more CD86 on the surface membrane. CREMα was found to bind to the CD86 promoter and suppressed its activity. Transfer of APCs from Crem−/− mice into naive mice facilitated a significantly stronger contact dermatitis response compared with mice into which APCs from Crem+/+ mice had been transferred. We conclude that CREMα is an important negative regulator of costimulation and APC-dependent T cell function both in vitro and in vivo.

Critical Role of Transcription Factor Cyclic AMP Response Element Modulator in β1-Adrenoceptor–Mediated Cardiac Dysfunction

Background— Chronic stimulation of the &bgr;1-adrenoceptor (&bgr;1AR) plays a crucial role in the pathogenesis of heart failure; however, underlying mechanisms remain to be elucidated. The regulation by transcription factors cAMP response element-binding protein (CREB) and cyclic AMP response element modulator (CREM) represents a fundamental mechanism of cyclic AMP–dependent gene control possibly implicated in &bgr;1AR-mediated cardiac deterioration. Methods and Results— We studied the role of CREM in &bgr;1AR-mediated cardiac effects, comparing transgenic mice with heart-directed expression of &bgr;1AR in the absence and presence of functional CREM. CREM inactivation protected from cardiomyocyte hypertrophy, fibrosis, and left ventricular dysfunction in &bgr;1AR-overexpressing mice. Transcriptome and proteome analysis revealed a set of predicted CREB/CREM target genes including the cardiac ryanodine receptor, tropomyosin 1&agr;, and cardiac &agr;-actin as altered on the mRNA or protein level along with the improved phenotype in CREM-deficient &bgr;1AR-transgenic hearts. Conclusions— The results imply the regulation of genes by CREM as an important mechanism of &bgr;1AR-induced cardiac damage in mice.

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Background: PP2A is a regulator of cardiac excitation-contraction coupling. Results: Cardiomyocyte-directed overexpression of B56α, the main cardiac PP2A regulatory subunit, results in the dephosphorylation of myofilament proteins, increased Ca2+ sensitivity, and higher contractility. Conclusion: This suggests an important role for B56α in regulating PP2A activity and thereby the contractile function. Significance: PP2A-B56α is a potential pharmacological target to improve cardiac performance in failing hearts. Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of −5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser16 was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca2+ sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.

CREB critically regulates action potential shape and duration in the adult mouse ventricle

The cAMP response element binding protein (CREB) belongs to the CREB/cAMP response element binding modulator/activating transcription factor 1 family of cAMP-dependent transcription factors mediating a regulation of gene transcription in response to cAMP. Chronic stimulation of β-adrenergic receptors and the cAMP-dependent signal transduction pathway by elevated plasma catecholamines play a central role in the pathogenesis of heart failure. Ion channel remodeling, particularly a decreased transient outward current (I(to)), and subsequent action potential (AP) prolongation are hallmarks of the failing heart. Here, we studied the role of CREB for ion channel regulation in mice with a cardiomyocyte-specific knockout of CREB (CREB KO). APs of CREB KO cardiomyocytes were prolonged with increased AP duration at 50 and 70% repolarization and accompanied by a by 51% reduction of I(to) peak amplitude as detected in voltage-clamp measurements. We observed a 29% reduction of Kcnd2/Kv4.2 mRNA in CREB KO cardiomyocytes mice while the other I(to)-related channel subunits Kv4.3 and KChIP2 were not different between groups. Accordingly, Kv4.2 protein was reduced by 37% in CREB KO. However, we were not able to detect a direct regulation of Kv4.2 by CREB. The I(to)-dependent AP prolongation went along with an increase of I(Na) and a decrease of I(Ca,L) associated with an upregulation of Scn8a/Nav1.6 and downregulation of Cacna1c/Cav1.2 mRNA in CREB KO cardiomyocytes. Our results from mice with cardiomyocyte-specific inactivation of CREB definitively indicate that CREB critically regulates the AP shape and duration in the mouse ventricle, which might have an impact on ion channel remodeling in situations of altered cAMP-dependent signaling like heart failure.

Protein phosphatase 2A is regulated by protein kinase Cα (PKCα)-dependent phosphorylation of its targeting subunit B56α at Ser41.

Background: PP2A activity and intracellular targeting are regulated by post-translational modifications of B56 phosphoprotein subunits. Results: PP2A is inhibited by a PKCα-dependent phosphorylation of B56α at Ser41 leading to downstream functional effects. Conclusion: This inhibition may represent an important signaling pathway regulated by stimuli that activate PKCα. Significance: Our data focus B56α on a dynamic role in the interplay between protein kinases and PP2A. Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser41 of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca2+ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser41. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca2+ homeostasis.

Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes

Chronic β-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to β-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the β-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca2+ releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca2+ waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca2+ transport and NCX1 protein level in TG. An increase in INCX and decrease in Ito and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.

Annexin A4 is a novel direct regulator of adenylyl cyclase type 5

Annexin A4 (AnxA4), a Ca2+‐ and phospholipid‐binding protein, is up‐regulated in the human failing heart In this study, we examined the impact of AnxA4 on β‐adrenoceptor (β‐AR)/cAMP‐dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK) 293 cells dose‐dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP‐Förster resonance energy transfer (EPAC‐FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162 vs. 1304 ± 185 fmol/μg protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart‐directed expression of the EPAC‐FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the β‐AR agonist isoproterenol (ISO) (anxA4a+/+ vs. anxA4a‐/‐: 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/μg protein) or FSK (anxA4a+/+ vs. anxA4a‐/‐: 1891 ± 238 vs. 2796 ± 343 fmol/μg protein; n = 9‐10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane‐bound AC type 5 (AC5). As a functional consequence of AnxA4‐mediated AC inhibition, AnxA4 inhibited the FSK‐induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10‐fold vs. control; n = 4 transfections) and reduced the FSK‐induced phosphorylation of the CRE‐binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 ± 17% vs. 105 ± 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)‐FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a‐/‐ mice was associated with an increased cardiac response to β‐AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.—Heinick, A., Husser, X., Himmler, K., Kirchhefer, U., Nunes, F., Schulte,J. S., Seidl, M. D., Rolfes, C., Dedman, J. R., Kaetzel, M. A., Gerke, V., Schmitz, W., Müller, F. U. Annexin A4 is a novel direct regulator of adenylyl cyclase type 5. FASEB J. 29, 3773‐3787 (2015). www.fasebj.org

A novel intronic promoter of the Crem gene induces small ICER (smICER) isoforms

The transcription factors cAMP‐responsive element binding protein (CREB) and cAMP‐responsive element modulator (CREM) regulate gene transcription in response to elevated cAMP levels. The Crem isoform inducible cAMP early repressor (Icer) is transcribed by the internal promoter P2 as a critical regulator of multiple cellular processes. Here, we describe a novel inducible Crem isoform, small Icer (smIcer), regulated by a newly identified promoter (P6). ChIP revealed binding of CREB to P6 in human and mouse myocardium. P6 activity was induced by constitutively active CREB or stimulation of adenylyl cyclase. In mice, smIcer mRNA was ubiquitously expressed and transiently induced by β‐adrenoceptor stimulation e.g., in heart and lung. SmICER repressed both basal and cAMP‐induced activities of P6 and P2 promoters. Stimulation of adenylyl cyclase induced P2 and P6 in cell type‐specific manner. Alternative translational start sites resulted in three different smICER proteins, linked to increased apoptosis sensitivity. In conclusion, the Crem gene provides two distinct and mutually controlled mechanisms of a cAMP‐dependent induction of transcriptional repressors. Our results suggest not only that smICER is a novel regulator of cAMP‐mediated gene regulation, but also emphasize that biological effects that have been ascribed solely to ICER, should be revised with regard to smICER.—Seidl, M. D., Nunes, F., Fels, B., Hildebrandt, I., Schmitz, W., Schulze‐Osthoff, K., and Müller, F. U. A novel intronic promoter of the Crem gene induces small ICER (smICER) isoforms. FASEB J. 28, 143–152 (2014). www.fasebj.org

Characterization of the Genetic Program Linked to the Development of Atrial Fibrillation in CREM-IbΔC-X Mice

Background— Reduced expression of genes regulated by the transcription factors CREB/CREM (cAMP response element-binding protein/modulator) is linked to atrial fibrillation (AF) susceptibility in patients. Cardiomyocyte-directed expression of the inhibitory CREM isoform CREM-Ib&Dgr;C-X in transgenic mice (TG) leads to spontaneous-onset AF preceded by atrial dilatation and conduction abnormalities. Here, we characterized the altered gene program linked to atrial remodeling and development of AF in CREM-TG mice. Methods and Results— Atria of young (TGy, before AF onset) and old (TGo, after AF onset) TG mice were investigated by mRNA microarray profiling in comparison with age-matched wild-type controls (WTy/WTo). Proteomic alterations were profiled in young mice (8 TGy versus 8 WTy). Annotation of differentially expressed genes revealed distinct differences in biological functions and pathways before and after onset of AF. Alterations in metabolic pathways, some linked to altered peroxisome proliferator–activated receptor signaling, muscle contraction, and ion transport were already present in TGy. Electron microscopy revealed significant loss of sarcomeres and mitochondria and increased collagen and glycogen deposition in TG mice. Alterations in electrophysiological pathways became prominent in TGo, concomitant with altered gene expression of K+-channel subunits and ion channel modulators, relevant in human AF. Conclusions— The most prominent alterations of the gene program linked to CREM-induced atrial remodeling were identified in the expression of genes related to structure, metabolism, contractility, and electric activity regulation, suggesting that CREM transgenic mice are a valuable experimental model for human AF pathophysiology.