Methods for comprehensive transcriptome analysis using next-generation sequencing and application in hypertrophic cardiomyopathy

Characterization of the RNA transcriptome by next-generation sequencing can produce an unprecedented yield of information that provides novel biologic insights. I describe four approaches for sequencing different aspects of the transcriptome and provide computational tools to analyze the resulting data. Methods that query the dynamic range of gene expression, low expressing transcripts, micro RNA levels, and start-site usage of transcripts are described

Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of $\sim$0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery–Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations

Barcoding bias in high-throughput multiplex sequencing of miRNA

Second-generation sequencing is gradually becoming the method of choice for miRNA detection and expression profiling. Given the relatively small number of miRNAs and improvements in DNA sequencing technology, studying miRNA expression profiles of multiple samples in a single flow cell lane becomes feasible. Multiplexing strategies require marking each miRNA library with a DNA barcode. Here we report that barcodes introduced through adapter ligation confer significant bias on miRNA expression profiles. This bias is much higher than the expected Poisson noise and masks significant expression differences between miRNA libraries. This bias can be eliminated by adding barcodes during PCR amplification of libraries. The accuracy of miRNA expression measurement in multiplexed experiments becomes a function of sample number

GO Analysis of Genes >2-fold Differentially Expressed Between Genotypes Unique to Specific Ligand Incubation Groups.

<p>GO Analysis of Genes >2-fold Differentially Expressed Between Genotypes Unique to Specific Ligand Incubation Groups.</p

<i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial RNA-seq datasets confirm cell identity and differential ligand response.

<p>(A) Cells express epicardial markers. Mean normalized reads between replicates and standard error are depicted. (B) Smooth muscle markers are markedly induced with TGFβ1 and TGFβ2 compared to BMP2 incubation. Fold is relative to VEH for each genotype. (C) Endothelial or myocardial markers are not expressed at significant levels (< 2 normalized reads). Mean normalized reads between replicates and standard error are depicted. (D) Genes >2-fold differentially expressed after ligand treatment compared to vehicle are depicted. Fewer genes are induced by incubation with TGFβ1–2 treatment in <i>Tgfbr3</i>^<i>-/-</i> epicardial cells compared to <i>Tgfbr3</i>^<i>+/+</i>, while the opposite is true with BMP incubation.</p

<i>Tgfbr3</i>^<i>-/-</i> epicardial cells fail to activate the NF-ĸB signaling pathway.

<p>(A) (TOP) Genes dysregulated in each group (>2-fold, p<0.001) were counted. (BOTTOM) Shared targets of NF-ĸB signaling dyregulated in all groups are shown. Red<b>—</b>expressed higher in<i>Tgfbr3</i>^<i>+/+</i>, Green<b>—</b>expressed higher in <i>Tgfbr3</i>^<i>-/-</i>. (B) Cells transfected with an NF-ĸB responsive SEAP reporter construct and incubated with VEH, TGFβ1, TGFβ2, or BMP2 revealed the inability of <i>Tgfbr3</i>^<i>-/-</i> cells to induce NF-ĸB signaling. (C) Incubation of <i>Tgfbr3</i>^<i>+/+</i> epicardial cells in a transwell invasion assay with an NF-ĸB inhibitor (BMS345541) significantly reduced invasion (* = p < .01) in response to ligands known to promote <i>Tgfbr3</i>-dependent invasion.</p

Cardiac-enriched BAF chromatin-remodeling complex subunit Baf60c regulates gene expression programs essential for heart development and function.

How chromatin-remodeling complexes modulate gene networks to control organ-specific properties is not well understood. For example, Baf60c (Smarcd3) encodes a cardiac-enriched subunit of the SWI/SNF-like BAF chromatin complex, but its role in heart development is not fully understood. We found that constitutive loss of Baf60c leads to embryonic cardiac hypoplasia and pronounced cardiac dysfunction. Conditional deletion of Baf60c in cardiomyocytes resulted in postnatal dilated cardiomyopathy with impaired contractile function. Baf60c regulates a gene expression program that includes genes encoding contractile proteins, modulators of sarcomere function, and cardiac metabolic genes. Many of the genes deregulated in Baf60c null embryos are targets of the MEF2/SRF co-factor Myocardin (MYOCD). In a yeast two-hybrid screen, we identified MYOCD as a BAF60c interacting factor; we showed that BAF60c and MYOCD directly and functionally interact. We conclude that Baf60c is essential for coordinating a program of gene expression that regulates the fundamental functional properties of cardiomyocytes

RNA-seq analysis identifies genes dysregulated in <i>Tgfbr3</i>^<i>-/-</i> epicardial cells.

<p>(A) (Left) The number of genes >2-fold (p<0.001) differentially expressed between <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial cells for each group. (Right) The number genes similarly dysregulated within selected groups that were also annotated in the IPA database are shown with genes found in each. (B) The number of overlapping genes >2-fold differentially regulated (p<0.001) was determined and mapped. 129 genes were similarly dysregulated across all groups. (C) (Top) Gene ontology analysis of these 129 genes by DAVID revealed a significant (p<0.0001) enrichment of genes associated with specific biological processes. emb.- embryonic. (Bottom) A representative network generated by gene regulatory network analysis of the 129 genes using Ingenuity Pathway Analysis software is depicted. Green- expressed higher in<i>Tgfbr3</i>^<i>+/+</i>, Red- expressed higher in <i>Tgfbr3</i>^<i>-/-</i>.</p