Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus.

BACKGROUND: Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS: Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS: We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSIONS: Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.

Genome-wide distribution of linker histone H1.0 is independent of MeCP2.

Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) of Flag-tagged-H1.0 in mouse forebrain excitatory neurons. Unexpectedly, Flag-H1.0 and MeCP2 occupied similar genomic regions and the Flag-H1.0 binding was not changed upon MeCP2 depletion. Furthermore, mild overexpression of H1.0 did not alter MeCP2 binding, suggesting that the functional binding of MeCP2 and H1.0 are largely independent.

An RNA interference screen identifies druggable regulators of MeCP2 stability.

Alterations in gene dosage due to copy number variation are associated with autism spectrum disorder, intellectual disability (ID), and other psychiatric disorders. The nervous system is so acutely sensitive to the dose of methyl-CpG-binding protein 2 (MeCP2) that even a twofold change in MeCP2 protein-either increased or decreased-results in distinct disorders with overlapping features including ID, autistic behavior, and severe motor dysfunction. Rett syndrome is caused by loss-of-function mutations in MECP2, whereas duplications spanning the MECP2 locus result in MECP2 duplication syndrome (MDS), which accounts for ~1% of X-linked ID. Despite evidence from mouse models that restoring MeCP2 can reverse the course of disease, there are currently no U.S. Food and Drug Administration-approved therapies available to clinically modulate MeCP2 abundance. We used a forward genetic screen against all known human kinases and phosphatases to identify druggable regulators of MeCP2 stability. Two putative modulators of MeCP2, HIPK2 (homeodomain-interacting protein kinase 2) and PP2A (protein phosphatase 2A), were validated as stabilizers of MeCP2 in vivo. Further, pharmacological inhibition of PP2A in vivo reduced MeCP2 in the nervous system and rescued both overexpression and motor abnormalities in a mouse model of MDS. Our findings reveal potential therapeutic targets for treating disorders of altered MECP2 dosage.

Maintenance of nucleosomal balance in cis by conserved AAA-ATPase Yta7.

The extent of chromatin compaction is a fundamental driver of nuclear metabolism . Yta7 is a chromatin-associated AAA-ATPase, the human ortholog of which, ANCCA/ATAD2 transcriptionally activates pathways of malignancy in a broad range of cancers. Yta7 directly binds histone H3, and bulk chromatin exhibits increased nucleosomal density in yta7Δ mutants. The suppression of yta7Δ mutant growth and transcriptional phenotypes in budding yeast by decreased dosage of histones H3 and H4 indicates the acute sensitivity of cells to deviations in nucleosome spacing. This study investigated the global changes in chromatin structure upon Yta7 loss or overexpression and determined which of these effects reflected direct Yta7 activity. Metagene analysis of Yta7's genome-wide localization indicated peak binding of Yta7 just downstream of the transcription start site. Cells lacking Yta7 exhibited increased nucleosome density within genes downstream of the +1 nucleosome, as defined by decreased internucleosomal distance, resulting in progressively 5'-shifted nucleosomes within the gene. In contrast, cells overexpressing Yta7 displayed profound 3'-shifts in nucleosome position and reduced nucleosome density within genes. Importantly, Yta7-bound regions were enriched for nucleosomal shifts, indicating that Yta7 acted locally to modulate nucleosome spacing. The phenotype of cells lacking both Yta7 and Rtt106, the histone H3/H4 chaperone, indicated that Yta7 functions in both Rtt106-dependent and Rtt106-independent ways to modulate nucleosome spacing within genes. This study suggested that Yta7 affected nucleosome density throughout the gene by both blocking Rtt106 from entering the gene, as shown previously at HTA1, and facilitating the loss of nucleosomes from the 5'-end.

Direct regulation of nucleosome density by the conserved AAA-ATPase Yta7.

Yta7 is a highly conserved bromodomain-containing protein with AAA-ATPase homology originally implicated in heterochromatin boundary function in Saccharomyces cerevisiae. Although increased activity of the human ortholog has been implicated in malignant breast tumors, Yta7's precise mode of action is unknown. Transcriptional analysis in yeast cells revealed a role for Yta7 and its ATPase function in gene induction, including galactose- and sporulation-induced transcription. This requirement was direct and activating, because Yta7 associated with the GAL gene cluster only upon transcriptional induction. Suggestive of a role in transcriptional elongation, Yta7 localized to the ORFs of highly transcribed genes. Intriguingly, the yta7Δ mutant's transcriptional defects were partially suppressed by decreased dosage of histones H3 and H4. Consistent with this suppression, cells lacking Yta7 exhibited both increased levels of chromatin-incorporated histone H3 and decreased nucleosome spacing. Importantly, this modulation of H3 levels occurred independently of changes in H3 transcript level. Because Yta7 binds histone H3 in vitro, these results suggested a direct role for Yta7 in H3 eviction or degradation. Further, local loss of Yta7 activity at a long inducible gene resulted in accumulation of H3 at the 3' end upon transcriptional activation, implying Yta7 may regulate H3 cotranscriptionally.

Circadian rhythms in Neurospora crassa: clock gene homologues in fungi.

Computer-based analysis of a total of 17 filamentous fungal and yeasts genomes has shown: (1) homologues of frq, wc-1, wc-2, and vvd, key gene components of the Neurospora crassa clock, are present in Magnaporthe grisea, Gibberella zeae, and Podospora anserina, suggesting an frq-based oscillator in these organisms; (2) some fungal species that are more distantly related to Neurospora, such as Rhizopus oryzae do not appear to have frq homologues; (3) many fungal species that do not appear to contain frq, such as Aspergillus nidulans, do contain wc homologues; (4) of 11 well-described genes classified as clock-controlled genes (ccgs), in Neurospora, all of them were found to have homologues in other fungi; (5) the ccg-8 gene of N. crassa has homologies to opi1p, a transcriptional regulatory gene in Saccharomyces cerevisiae involved in inositol regulation. This suggests the possibilities of rhythmic inositol regulation, and/or a cascade of rhythmic activation of other genes in N. crassa.