DOT1L regulates dystrophin expression and is critical for cardiac function

Abstract

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin-glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.

Figure 1.

Disruption of DOT1L function in mouse cardiomyocytes results in heart dilation and lethality. (A) Dot1L CKO in mouse heart causes postnatal and adult lethality. Survival curves of wild-type (WT), HET, and CKO mice. Fifty percent of CKO mice die within the first 2 wk after birth and the remaining 50% die by 6 mo of age. (B) CKO hearts are severely enlarged compared with wild type (WT). Shown are wild-type and CKO hearts harvested from P10 and 5 -mo-old adult mice, respectively. Bar, 1 mm. (C) H&E staining of paraffin tissue sections indicated that CKO hearts are enlarged due to ventricular chamber dilation. Bar, 1 mm. (D) CKO mice have increased heart to body weight ratios. Heart weight (milligrams) to body weight (grams) ratios were calculated using an analytical balance. CKO mice have increased ratios compared with wild-type littermates. (*) P < 0.06; (**) P < 0.006; (***) P < 0.0006. (E) CKO mice have increased heart weight. Heart weight (milligrams) was measured using an analytical balance. (*) P < 0.06.

Figure 2.

Disruption of DOT1L function in mouse cardiomyocytes results in pathologic cardiac remodeling. (A) Positive TUNEL staining (green) merged with DAPI (blue) demonstrates increased cell death in CKO hearts (P10; bar, 5 μm). (B) TEM analysis of P10 hearts demonstrates increased autophagic cell death (cf. panels i and ii; yellow box is enlarged from panel ii; vacuoles are indicated by yellow arrows; bar, 2 μm) and myofibroblast infiltration (indicated by asterisk [*] in panel iii) in CKO hearts. Note that myocytes in wild-type (WT) hearts show tight lateral association, whereas myocytes in CKO hearts have large gaps due to myofibroblast infiltration. (C) Increased interstitial ECM, indicated by staining of the ECM component HSPG2 (red), is observed in the CKO hearts (arrowheads). Increased ECM and myofibroblasts lining the inner left ventricular chamber wall (between the two yellow lines) is also observed in CKO hearts. Bar, 10 μm. (D) Masson's trichrome staining of paraffin tissue sections of mouse hearts (5 mo old). Two enlarged regions are shown (black and yellow boxes). Interstitial fibrosis is seen in CKO hearts, but not in wild-type (WT) counterparts. Bar, 5 mm. (E) RT-qPCR analysis demonstrates activation of fetal-specific genes (Myh7, Acta1, Nppa, and Nppb) in the CKO hearts. In contrast, down-regulation of adult Myh6 is also observed. (F) Increased cell proliferation in CKO hearts (P1 and P5). Frozen tissue sections were stained with anti-Ki-67, a marker of cell proliferation, and counterstained with DAPI. The percentage of proliferating cells was calculated by dividing the number of Ki-67-positive nuclei by total nuclei and multiplying by 100.

Figure 3.

Disruption of DOT1L function in mouse cardiomyocytes results in conduction abnormalities. (A) Representative EKGs for wild-type (Wt) and CKO mice. The analysis was performed using 5-mo-old mice (n = 8 per group). CKOa has complete AV dissociation, as evidence by nonsustained ventricular tachycardia, while CKOb has a Type II second-degree heart block. Bar, 200 msec. (B–E) Quantification of EKG data indicated an overall significant increase in RR interval (B), PR interval (C), P-wave duration (D), and QRS interval (E). P-values were calculated by Student t-test.

Figure 4.

Dystrophin is a direct target of DOT1L. (A) RT-qPCR analysis using RNAs isolated from P10 wild-type (WT) and CKO hearts (n = 12). Dmd and Ttn expression is down-regulated in Dot1L CKO hearts. Other members of the DGC (Dag1, Sgca, Sgcb, Sgcd, and Sgcg), as well as selected DCM causal genes (Actn2, Ldb3, Des, and Taz), remain unchanged. (B,C) Immunostaining of frozen heart sections demonstrated loss of Dmd protein in CKO hearts (green). Consistent with a Dmd deficiency and complex instability, reduction of βDG (B) and SGCA (C) is observed. (D) Micro-ChIP using heart tissues from P10 pups demonstrates that H3K79me2/3 is enriched in the gene body of Dmd, and the enrichment is dependent on functional DOT1L. Amplicon #1 located ∼15 kb upstream of the TSS serves as background for H3K79me2/3 enrichment. (E) ChIP using an anti-SRF antibody demonstrates that SRF binding to the CArG-box consensus region of the Dmd muscle-specific promoter is not affected in Dot1L CKO hearts. Amplification at the −15-kb TSS serves as a background for SRF enrichment. (F) RT-qPCR analysis demonstrates that Dot1L KD in C2C12 cells results in down-regulation of Dmd. Error bars represent SD of three independent experiments. (G,H) ChIP analysis demonstrates binding of F-DOT1L to the Dmd locus, and its methylation on H3K79 is dependent on DOT1L enzymatic activity.

Figure 5.

Rescue of electrical conduction in CKO mice by expression of minidystrophin gene. (A–D) The defective function of the CKO heart can be rescued by expression of a minidystrophin gene when injected at P3 and analyzed at 5 mo of age, as indicated by the lack of significant difference between wild-type (WT) and CKO rescued mice. (E–H) Additionally, minidystrophin can rescue adult CKO mice injected at 2 mo in terms of RR interval (E), P-wave duration (G), and QRS interval (H). (F) In addition, PR interval was partially rescued, as the difference observed is less than without miniDmd. (P = 0.02 with rescue vs. P = 0.00014 [Fig. 3C] without rescue at 5 mo). (E,F) At 8 mo of age, past the second stage of lethality, CKO + miniDmd mice still maintain similar rescued levels of electrical conduction performance.