Adiponectin is one of the most abundant circulating hormones, which through adenosine monophosphate-activated protein kinase (AMPK), enhances fatty acid and glucose oxidation, and exerts a cardioprotective effect. However, its effects on cellular bioenergetics have not been explored. We have previously reported that 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, an AMPK activator) enhances mitochondrial respiration through a succinate dehydrogenase (SDH or complex II)-dependent mechanism in cardiac myocytes, leading us to predict that Adiponectin would exert a similar effect via activating AMPK. Our results show that Adiponectin enhances basal mitochondrial oxygen consumption rate (OCR), ATP production, and spare respiratory capacity (SRC), which were all abolished by the knockdown of AMPKγ1, inhibition of SDH complex assembly, via the knockdown of the SDH assembly factor 1 (Sdhaf1), or inhibition of SDH activity. Additionally, Adiponectin alleviated hypoxia-induced reductions in OCR and ATP production, in a Sdhaf1-dependent manner, whereas overexpression of Sdhaf1 confirmed its sufficiency for mediating these effects. Importantly, the levels of holoenzyme SDH under the various conditions correlated with OCR. We also show that the effects of Adiponectin, AMPK, Sdhaf1, as well as, SDH complex assembly all required sirtuin 3 (Sirt3). In conclusion, Adiponectin potentiates mitochondrial bioenergetics via promoting SDH complex assembly in an AMPK-, Sdhaf1-, and Sirt3-dependent fashion in cardiac myocytes.

H2A.Z plays a fundamental role in the regulation of transcription and epigenetics, however, the mechanisms that underlie its functions are not fully understood. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with an array of tricarboxylic acid cycle and beta-oxidation enzymes in the mouse heart. Recombinant green florescence fusion proteins combined with mutations of putative nuclear localization signals of select enzymes, including acetyl-CoA acyltransferase 2 (ACAA2), oxoglutarate dehydrogenase (OGDH), and isocitrate dehydrogenase 2 confirmed their nuclear localization and chromatin binding in both rodent and human cells. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the selective association of ACAA2 and OGDH with H2A.Z-occupied transcription start sites. Finally, human H2A.Z-deficient HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, with the exception of pyruvate dehydrogenase, accompanied with reduced posttranslational histone modifications. Thus, the data show that metabolic enzymes are recruited to active promoters for potential site-directed epigenetic modifications.

Histone H2A.Z plays an essential role in regulating transcriptional rates and memory. Interestingly, H2A.Z-bound nucleosomes are located in both transcriptionally active and inactive promotors, with no clear understanding of the mechanisms via which it differentially regulates transcription. We hypothesized that its functions are mediated through recruitment of regulatory proteins to promoters. Using rapid chromatin immunoprecipitation-mass spectrometry, we uncovered the association of H2A.Z-bound chromatin with the metabolic enzymes, oxoglutarate dehydrogenase (OGDH) and acetyl-CoA acyltransferase 2 (ACAA2). Recombinant green florescence fusion proteins, combined with mutations of predicted nuclear localization signals, confirmed their nuclear localization and chromatin binding. Conclusively, chromatin immunoprecipitation-deep sequencing, confirmed the predominant association of OGDH and ACAA2 with H2A.Z-occupied transcription start sites and enhancers, the former of which we confirmed is conserved in both mouse and human tissue. Furthermore, H2A.Z-deficient human HAP1 cells exhibited reduced chromatin-bound metabolic enzymes, accompanied with reduced posttranslational histone modifications, including acetylation and succinylation. Specifically, knockdown of OGDH diminished H4 succinylation. Thus, the data reveal that select metabolic enzymes are assembled at active, H2A.Z-occupied, promoters, for potential site-directed production of metabolic intermediates that are required for histone modifications.

The mechanisms that regulate H2A.Z and its requirement for transcription in differentiated mammalian cells remains ambiguous. In this study, we identified the interaction between the C-terminus of ANP32e and N-terminus of H2A.Z in a yeast two-hybrid screen. Knockdown of ANP32e resulted in proteasomal degradation and nuclear depletion of H2A.Z or of a chimeric green florescence protein fused to its N-terminus. This effect was reversed by inhibition of protein phosphatase 2A (PP2A) and, conversely, reproduced by overexpression of its catalytic subunit. Accordingly, knockdown of ANP32e inhibited phosphorylation of H2A.Z, whereas a mutation of serine-9 proved its requirement for both the protein's stability and nuclear localization, as did knockdown of the nuclear mitogen and stress-induced kinase 1. Moreover, ANP32e's knockdown also revealed its differential requirement for cell signaling and gene expression, whereas, genome-wide binding analysis confirmed its co-localization with H2A.Z at transcription start sites, as well as, gene bodies of inducible and tissue-specific genes. The data also suggest that H2A.Z restricts transcription, which is moderated by ANP32e at the promoter and gene bodies of expressed genes. Thus, ANP32e, through inhibition of PP2A, is required for nucleosomal inclusion of H2A.Z and the regulation of gene expression.

Cardiac hypertrophy is a manifestation of an increase in the workload imposed on the heart. The cause, duration, and extent of the workload dictate the physiological vs. pathological nature of the hypertrophy and its likelihood to transition into cardiac failure. Our goal is to identify differences between these 2 forms of hypertrophy by examining the binding patterns of key basic transcription factors and regulators in a genome-wide fashion. Our data show that a pause-release mechanism of RNA polymerase II (pol II) bound to the transcriptional start site (TSS) induces housekeeping/essential genes (~ 63% of expressed genes), in a synchronous and incremental fashion that is proportional to the increase in myocyte size and does not require de novo pol II binding. This mechanism is responsible for the increase in cell size and mass that constitute hypertrophy in both physiological and pathological forms. Superimposed on this is a second and critical group of genes (~3.5%) that are stress-induced in the pathological form of hypertrophy but are minimally, if at all, expressed in the postnatal and adult heart (e.g. Acta1, Ankrd1, Xirp2, Nppa, Col1a1 ..etc). In contrast to the housekeeping genes, these genes require de novo pol II recruitment and exhibit a robust increase in expression. To further identify the transcriptional mechanisms that distinguish these sets of genes, we performed chromatin immunoprecipitation-deep sequencing (ChIP-Seq) analysis for cyclin-dependent kinase 9 (Cdk9) and histone H2A.z. The results reveal an increase in Cdk9 at the TSS of paused genes vs. an increase at TSS and throughout the gene body of inducible genes. In contrast, H2A.z levels remained constant. However, we found that Acidic Nuclear Protein 32e (Anp32e), a nuclear protein phosphatase 2A (PP2A) inhibitor, interacted with H2A.z and regulated phosphorylation of Cdk9. Interestingly, knockdown of Anp32e during pressure overload hypertrophy resulted in 70-90% inhibition of stress-inducible genes, but only an incremental inhibition of the increase in cardiac size. Thus, we propose that targeting Anp32e may partially revert pathological hypertrophy to a more physiological form via specifically inhibiting the stress-induced genes while preserving the increase in cardiac mass.

Genome-wide modifications in basic transcriptional activity underlie cardiac development and disease. We have previously reported that transcriptional regulation by RNA polymerase II (pol II) distinguishes different functional gene categories. In particular, transcription of constitutive, housekeeping, genes is regulated by a pause/release mechanism vs. de novo recruitment of pol II to cardiac-specific and inducible genes during cardiac hypertrophy in mice. Our objective is to identify the epigenetics and nuclear proteins that distinguish these transcriptional patterns. Using chromatin immunoprecipitation-deep sequencing (ChIP-Seq) we identified relatively high levels of H2A.z at the transcriptional start site of all housekeeping and inducible genes that exhibited only minimal or no changes in the heart before or after induction of pressure overload. In contrast, however, all cardiac-specific genes were consistently devoid of H2A.z. We also identified the specific interaction between H2A.z and acidic nuclear protein 32e (ANP32e), which inhibits the dephosphorylation and deactivation of cyclin-dependent kinase 9 (Cdk9) by protein phosphatase 2A (PP2A) and enhances its recruitment to chromatin. Accordingly, cardiac-specific and inducible genes diverge with regards to their regulation by Cdk9, where pressure overload induces a strict decrease vs. increase of Cdk9, respectively, at the promoter and gene body regions of these gene categories. Paradoxically, the reduction in Cdk9 in the former genes is accompanied by an increase in pol II levels, with a net result of little or no change in transcriptional rates. On the other hand, inducible genes are characterized by high levels of de novo recruitment of both pol II and Cdk9, with a sharp increase in transcriptional rates, which was specifically and completely inhibited by knockdown of Cdk9, or its inhibitors 5, 6- dichloro-1-β-D-ribofuranosyl-1H-benzimidazole or inhibitor II. Thus, the differential recruitment of H2A.z and Cdk9 determines the elongational rate of pol II that discriminates between the transcriptional rates of pressure overload-induced v. constitutively-expressed cardiac-specific genes.

Hypoxia-inducible factor 1α (Hif-1α) is the primary transcription factor that promptly increases upon exposure of the cells to hypoxia. It is mainly regulated by posttranscriptional and posttranslational mechanisms involving miR-199a and prolyl hydroxylase, respectively. In addition, the discovery of multiple Hif-1α isoforms suggests that alternative splicing may play a role in regulating its activity, although little is known about this in the heart. We, thus, hypothesized, that in addition to the known mechanisms, Hif-1α is activated by an alternative splicing step, a process that is tightly coupled to transcription. To test this, we treated myocytes with antimiR-199a, which robustly induces Hif-1α expression, and analyzed its effects on gene transcription using RNA polymerase II Chromatin immunoprecipitation-deep sequencing. The analysis revealed that antimiR-199a induced 1) the upregulation of genes that are predominantly Hif-1α targets including Pdk1, Glut3, Vegfa, Bnip3, Ndrg1, and Gys1, in addition to Pfkm, G0s2 and Sdpr, which are a subject of an independent study, and 2) a unique, very precise RNA pol II pausing (accumulation of pol II at a very precise sites with a density >2x of its flanking sequence) within the exons of only the Hif-1α gene, while RNA pol II at the transcription start site, and within other regions of the gene body, was similar to the control myocytes. In particular, all exons except for exons 1, 10, and 11, exhibited some degree of pausing, where exon 15 had a pol II pausing peak that was 20 fold higher than the flanking pol II density. Deceleration of transcriptional elongation has been associated with RNA splicing, however, these very precise exonic pausing peaks are quite unique. Using quantitative PCR, were able to confirm that during normoxia, Hif-1α was abundant as determined by primers specific for exons 10, 11, and 12, which increased by ~2 folds with miR-199a treatment. On the other hand, exon 15, which encodes part of the transactivation domain, was relatively lower during normoxia but increased 300x with antimiR-199a treatment. The result suggests that alternative splicing of exon 15 is a critical regulatory step required for the transcriptional activity of Hif-1α, which is associated with the increase in its protein.