5′RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy
Danos C. Christodoulou, Hiroko Wakimoto, Kenji Onoue, Seda Eminaga, Joshua M. Gorham, Steve R. DePalma, Daniel S. Herman, Polakit Teekakirikul, David A. Conner, David M. McKean, Andrea A. Domenighetti, Anton Aboukhalil, Stephen Chang, Gyan Srivastava, Barbara McDonough, Philip L. De Jager, Ju Chen, Martha L. Bulyk, Jochen D. Muehlschlegel, Christine E. Seidman, J.G. Seidman
Danos C. Christodoulou, Hiroko Wakimoto, Kenji Onoue, Seda Eminaga, Joshua M. Gorham, Steve R. DePalma, Daniel S. Herman, Polakit Teekakirikul, David A. Conner, David M. McKean, Andrea A. Domenighetti, Anton Aboukhalil, Stephen Chang, Gyan Srivastava, Barbara McDonough, Philip L. De Jager, Ju Chen, Martha L. Bulyk, Jochen D. Muehlschlegel, Christine E. Seidman, J.G. Seidman
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Technical Advance Cardiology

5′RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy

Abstract

The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5′ start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5′ start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5′RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X–encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5′RNA-Seq has the potential to identify genome-wide changes in 5′ start-site usage that are associated with pathogenic phenotypes.

Authors

Danos C. Christodoulou, Hiroko Wakimoto, Kenji Onoue, Seda Eminaga, Joshua M. Gorham, Steve R. DePalma, Daniel S. Herman, Polakit Teekakirikul, David A. Conner, David M. McKean, Andrea A. Domenighetti, Anton Aboukhalil, Stephen Chang, Gyan Srivastava, Barbara McDonough, Philip L. De Jager, Ju Chen, Martha L. Bulyk, Jochen D. Muehlschlegel, Christine E. Seidman, J.G. Seidman

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Figure 2

Fhl1 transcripts in WT and MHC403/+ LV and isolated myocytes and nonmyocytes.

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Fhl1 transcripts in WT and MHC403/+ LV and isolated myocytes and nonmyo...
(A) UCSC genome browser screen shot of the Fhl1 gene, showing normalized read depths found in WT and HCM (MHC403/+) hearts. Structures of bFhl1 and iFhl1 isoforms are shown. Note the 10-fold difference in y axis scales; iFhl1 in HCM hearts was expressed at a 10-fold higher level than bFhl1 in WT hearts. (B) Fhl1 5′RACE products fractionated on an agarose gel confirmed 5′RNA-Seq estimates of bFHL1 and iFHL1 levels. For analyses of PCR products, see Supplemental Figure 7. (C) Western blot showing increased iFHL1 levels in HCM LV protein extracts. Extracts (15 μg Fhl1-null, 15 μg WT, 5 μg MHC403/+ per lane) were fractionated on a 12% SDS-polyacrylamide gel. Note the size shift due to the addition of 16 amino acids to the iFHL1 protein (∼1.6 kDa) compared with the bFHL1 protein. (D) iFhl1 and bFhl1 expression in LV, isolated myocytes, and nonmyocytes from 3 WT and 3 MHC403/+ mice. No iFhl1 transcripts were detected in nonmyocytes from WT or MHC403/+ hearts. Reads were normalized to total reads per million (see Methods). *P < 5 × 10–6, **P < 10–7, ***P = 2 × 10–16, Fisher exact test.