Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation.

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

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SUMMARY High energy production in mitochondria is essential for maintaining cardiac contraction in the heart. Genes regulating mitochondrial function are commonly downregulated during heart failure. Here we show that both PPARα and Sirt1 are upregulated by pressure overload in the heart. Haploinsufficiency of either PPARα or Sirt1 attenuated pressure overload-induced cardiac hypertrophy and failure, whereas simultaneous upregulation of PPARα and Sirt1 exacerbated the cardiac dysfunction. PPARα and Sirt1 coordinately suppressed genes involved in mitochondrial function that are regulated by estrogen related receptors (ERRs). PPARα bound and recruited Sirt1 to the ERR response element (ERRE), thereby suppressing ERR target genes in an RXR-independent manner. Downregulation of ERR target genes was also observed during fasting, and this appeared to be an adaptive response of the heart. These results suggest that suppression of the ERR transcriptional pathway by PPARα/Sirt1, a physiological fasting response, is involved in the progression of heart failure by promoting mitochondrial dysfunction.

Rationale Oxidation of cysteine residues in class II histone deacetylases (HDACs), including HDAC4, causes nuclear exit, thereby inducing cardiac hypertrophy. The cellular source of reactive oxygen species (ROS) responsible for oxidation of HDAC4 remains unknown. Objective We investigated whether Nox4, a major NADPH oxidase, mediates cysteine oxidation of HDAC4. Methods and Results Phenylephrine (PE, 100 μM), an α1 adrenergic agonist, induced upregulation of Nox4 (1.5-fold, p<0.05) within 5 min, accompanied by increases in O2− (3.5-fold, p<0.01) from the nuclear membrane and nuclear exit of HDAC4 in cardiomyocytes (CM). Knockdown of Nox4, but not Nox2, attenuated O2− production in the nucleus and prevented PE-induced oxidation and nuclear exit of HDAC4. After continuous infusion of PE (20 mg/kg/day) for 14 days, wild-type (WT) and cardiac-specific Nox4 knockout (c-Nox4 KO) mice exhibited similar aortic pressures. Left ventricular (LV) weight/tibial length (5.7 ±0.2 vs. 6.4 ±0.2 mg/mm, p<0.05) and CM cross-sectional area (223 ±13 vs. 258 ±12 μm2, p<0.05) were significantly smaller in c-Nox4 KO than in WT mice. Nuclear O2− production in the heart was significantly lower in c-Nox4 KO than in WT mice (4116 ±314 vs. 7057 ±1710 RLU, p<0.05), and cysteine oxidation of HDAC4 was decreased. HDAC4 oxidation and cardiac hypertrophy were also attenuated in c-Nox4 KO mice 2 weeks after transverse aortic constriction. Conclusions Nox4 plays an essential role in mediating cysteine oxidation and nuclear exit of HDAC4, thereby mediating cardiac hypertrophy in response to PE and pressure overload.