Mark Ziemann

Multicellular transcriptional analysis of mammalian heart regeneration

Background: The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we assemble a transcriptomic framework of multiple cardiac cell populations during postnatal development and following injury, which enables comparative analyses of the regenerative (neonatal) versus nonregenerative (adult) state for the first time. Methods: Cardiomyocytes, fibroblasts, leukocytes, and endothelial cells from infarcted and noninfarcted neonatal (P1) and adult (P56) mouse hearts were isolated by enzymatic dissociation and fluorescence-activated cell sorting at day 3 following surgery. RNA sequencing was performed on these cell populations to generate the transcriptome of the major cardiac cell populations during cardiac development, repair, and regeneration. To complement our transcriptomic data, we also surveyed the epigenetic landscape of cardiomyocytes during postnatal maturation by performing deep sequencing of accessible chromatin regions by using the Assay for Transposase-Accessible Chromatin from purified mouse cardiomyocyte nuclei (P1, P14, and P56). Results: Profiling of cardiomyocyte and nonmyocyte transcriptional programs uncovered several injury-responsive genes across regenerative and nonregenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts were characterized by the expression of a proliferative gene expression network following infarction, which mirrored the neonatal state. In contrast, cardiomyocytes failed to reactivate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during postnatal maturation. Conclusions: This work provides a comprehensive framework and transcriptional resource of multiple cardiac cell populations during cardiac development, repair, and regeneration. Our findings define a regulatory program underpinning the neonatal regenerative state and identify alterations in the chromatin landscape that could limit reinduction of the regenerative program in adult cardiomyocytes.

Endothelial Transcriptome in Response to Pharmacological Methyltransferase Inhibition

The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non‐histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7′‐carbonylbis (azanediyl) bis(4‐hydroxynaphthalene‐2 ‐sulfonic acid (AMI‐1) and disodium‐2‐(2,4,5,7‐ tetrabromo‐3‐oxido‐6‐oxoxanthen‐9‐yl) benzoate trihydrate (AMI‐5). Deep sequencing of mRNA indicated robust change on transcription following AMI‐5 treatment compared with AMI‐1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI‐5 but not AMI‐1 was found to decrease methylation of H3 histones at lysineu20054 and down‐regulate gene expression associated with interleukin‐6 (IL‐6) and activator protein‐1 (AP‐1) signaling pathways. These results imply that inhibition of protein methylation by AMI‐1 and AMI‐5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium.

RAGE Deletion Confers Renoprotection by Reducing Responsiveness to Transforming Growth Factor-β and Increasing Resistance to Apoptosis

Signaling via the receptor of advanced glycation end products (RAGE)—though complex and not fully elucidated in the setting of diabetes—is considered a key injurious pathway in the development of diabetic nephropathy (DN). We report here that RAGE deletion resulted in increased expression of fibrotic markers (collagen I and IV, fibronectin) and the inflammatory marker MCP-1 in primary mouse mesangial cells (MCs) and in kidney cortex. RNA sequencing analysis in MCs from RAGE−/− and wild-type mice confirmed these observations. Nevertheless, despite these gene expression changes, decreased responsiveness to transforming growth factor-β was identified in RAGE−/− mice. Furthermore, RAGE deletion conferred a more proliferative phenotype in MCs and reduced susceptibility to staurosporine-induced apoptosis. RAGE restoration experiments in RAGE−/− MCs largely reversed these gene expression changes, resulting in reduced expression of fibrotic and inflammatory markers. This study highlights that protection against DN in RAGE knockout mice is likely to be due in part to the decreased responsiveness to growth factor stimulation and an antiapoptotic phenotype in MCs. Furthermore, it extends our understanding of the role of RAGE in the progression of DN, as RAGE seems to play a key role in modulating the sensitivity of the kidney to injurious stimuli such as prosclerotic cytokines.

Transcription factors Tp73 , Cebpd , Pax6, and Spi1 rather than DNA methylation regulate chronic transcriptomics changes after experimental traumatic brain injury

Traumatic brain injury (TBI) induces a wide variety of cellular and molecular changes that can continue for days to weeks to months, leading to functional impairments. Currently, there are no pharmacotherapies in clinical use that favorably modify the post-TBI outcome, due in part to limited understanding of the mechanisms of TBI-induced pathologies. Our system biology analysis tested the hypothesis that chronic transcriptomics changes induced by TBI are controlled by altered DNA-methylation in gene promoter areas or by transcription factors. We performed genome-wide methyl binding domain (MBD)-sequencing (seq) and RNA-seq in perilesional, thalamic, and hippocampal tissue sampled at 3xa0months after TBI induced by lateral fluid percussion in adult male Sprague-Dawley rats. We investigated the regulated molecular networks and mechanisms underlying the chronic regulation, particularly DNA methylation and transcription factors. Finally, we identified compounds that modulate the transcriptomics changes and could be repurposed to improve recovery. Unexpectedly, DNA methylation was not a major regulator of chronic post-TBI transcriptomics changes. On the other hand, the transcription factors Cebpd, Pax6, Spi1, and Tp73 were upregulated at 3xa0months after TBI (False discovery rateu2009<u20090.05), which was validated using digital droplet polymerase chain reaction. Transcription regulatory network analysis revealed that these transcription factors regulate apoptosis, inflammation, and microglia, which are well-known contributors to secondary damage after TBI.xa0Library of Integrated Network-based Cellular Signatures (LINCS) analysis identified 118 pharmacotherapies that regulate the expression of Cebpd, Pax6, Spi1, and Tp73. Of these, the antidepressant and/or antipsychotic compounds trimipramine, rolipramine, fluspirilene, and chlorpromazine, as well as the anti-cancer therapies pimasertib, tamoxifen, and vorinostat were strong regulators of the identified transcription factors, suggesting their potential to modulate the regulated transcriptomics networks to improve post-TBI recovery.

MeCP2 interacts with chromosomal microRNAs in brain

ABSTRACT Although methyl CpG binding domain protein-2 (MeCP2) is commonly understood to function as a silencing factor at methylated DNA sequences, recent studies also show that MeCP2 can bind unmethylated sequences and coordinate gene activation. MeCP2 displays broad binding patterns throughout the genome, with high expression levels similar to histone H1 in neurons. Despite its significant presence in the brain, only subtle gene expression changes occur in the absence of MeCP2. This may reflect a more complex regulatory mechanism of MeCP2 to complement chromatin binding. Using an RNA immunoprecipitation of native chromatin technique, we identify MeCP2 interacting microRNAs in mouse primary cortical neurons. In addition, comparison with mRNA sequencing data from Mecp2-null mice suggests that differentially expressed genes may indeed be targeted by MeCP2-interacting microRNAs. These findings highlight the MeCP2 interaction with microRNAs that may modulate its binding with chromatin and regulate gene expression.

Valproic acid attenuates hyperglycemia-induced complement and coagulation cascade gene expression

Atherothrombosis remains the leading cause of morbidity and mortality in patients diagnosed with diabetes mellitus, but the molecular mechanisms underpinning this remain unresolved. As the liver plays a major role in metabolic homeostasis and secretion of clotting factors and inflammatory innate immune proteins, there is an interest in understanding the mechanisms of hepatic cell activation under hyperglycemia and whether this can be attenuated pharmacologically. We have previously shown that hyperglycemia stimulates major changes in chromatin organisation and metabolism in hepatocytes, and that the histone deacetylase inhibitor valproic acid (VPA; IUPAC: 2-propylpentanoic acid) is able to reverse some of these metabolic changes. In this study, we used deep transcriptome sequencing to show that VPA attenuates hyperglycemia-induced activation of complement and coagulation cascade genes. These findings reveal a novel mechanism of VPA protection against hyperglycemia, which might improve the therapeutic approaches for diabetes.

Diet during Pregnancy is Implicated in the Regulation of Hypothalamic RNA Methylation and Risk of Obesity in Offspring

SCOPEnEarly life nutrition has long-lasting influence in adults through key mediators that modulate epigenetic states, although the determinants involved that underlie this response remain controversial. Because of the similarities between metabolic, physiological, and endocrine changes and those occurring in human type 2 diabetes, we studied the interaction of diet during pregnancy regulating RNA adenosine methylation (N6-methyladenosine [m6A]) and the transcriptome in Psammomys obesus.nnnMETHODS AND RESULTSnBreeding pairs were randomly allocated standard diet (total digestible energy 18xa0MJxa0kg-1 ) or low-fat diet (15xa0MJxa0kg-1 ). Offspring were weaned onto the low-fat diet at 4 weeks of age and given ad libitum access, resulting in two experimental groups: 1) male offspring of animals fed a low-fat diet and weaned onto the low-fat diet and 2) male offspring of animals fed a standard diet and weaned onto the low-fat diet. Hypothalamic RNA was used to assess m6A by immunoprecipitation. Parental low-fat diet alters the metabolic phenotype in offspring. An association between parental diet and hypothalamic m6A was observed in regulating the expression of FTO and METTL3 in the offspring.nnnCONCLUSIONSnWe propose the regulatory capacity is now broadened for the first time to include m6A in developmental programming and obesity phenotype.

Age-Related Differential Structural and Transcriptomic Responses in the Hypertensive Heart

While aging is a critical risk factor for heart failure, it remains uncertain whether the aging heart responds differentially to a hypertensive stimuli. Here we investigated phenotypic and transcriptomic differences between the young and aging heart using a mineralocorticoid-excess model of hypertension. Ten-week (“young”) and 36-week (“aging”) mice underwent a unilateral uninephrectomy with deoxycorticosterone acetate (DOCA) pellet implantation (n = 6–8/group) and were followed for 6 weeks. Cardiac structure and function, blood pressure (BP) and the cardiac transcriptome were subsequently examined. Young and aging DOCA mice had high BP, increased cardiac mass, cardiac hypertrophy, and fibrosis. Left ventricular end-diastolic pressure increased in aging DOCA-treated mice in contrast to young DOCA mice. Interstitial and perivascular fibrosis occurred in response to DOCA, but perivascular fibrosis was greater in aging mice. Transcriptomic analysis showed that young mice had features of higher oxidative stress, likely due to activation of the respiratory electron transport chain. In contrast, aging mice showed up-regulation of collagen formation in association with activation of innate immunity together with markers of inflammation including cytokine and platelet signaling. In comparison to younger mice, aging mice demonstrated different phenotypic and molecular responses to hypertensive stress. These findings have potential implications for the pathogenesis of age-related forms of cardiovascular disease, particularly heart failure.

Systems approach to the pharmacological actions of HDAC inhibitors reveals EP300 activities and convergent mechanisms of regulation in diabetes

ABSTRACT Given the skyrocketing costs to develop new drugs, repositioning of approved drugs, such as histone deacetylase (HDAC) inhibitors, may be a promising strategy to develop novel therapies. However, a gap exists in the understanding and advancement of these agents to meaningful translation for which new indications may emerge. To address this, we performed systems-level analyses of 33 independent HDAC inhibitor microarray studies. Based on network analysis, we identified enrichment for pathways implicated in metabolic syndrome and diabetes (insulin receptor signaling, lipid metabolism, immunity and trafficking). Integration with ENCODE ChIP-seq datasets identified suppression of EP300 target genes implicated in diabetes. Experimental validation indicates reversal of diabetes-associated EP300 target genes in primary vascular endothelial cells derived from a diabetic individual following inhibition of HDACs (by SAHA), EP300, or EP300 knockdown. Our computational systems biology approach provides an adaptable framework for the prediction of novel therapeutics for existing disease.

Accuracy, speed and error tolerance of short DNA sequence aligners

Aligning short DNA sequence reads to the genome is an early step in the processing of many types of genomics data, and impacts on the fidelity of downstream results. In this work, the accuracy, speed and tolerance to errors are evaluated in read of varied length for six commonly used mapping tools; BWA aln, BWA mem, Bowtie2, Soap2, Subread and STAR. The accuracy evaluation using Illumina-like simulated reads showed that accuracy varies by read length, but overall BWA aln was most accurate, followed by BWA mem and Bowtie2. BWA mem was most accurate with Ion Torrent-like read sets. STAR was at least 5 fold faster than Bowtie2 or BWA mem. BWA mem tolerated the highest density of mismatches and indels compared to other mappers. These data provide important accuracy and speed benchmarks for commonly used mapping software.