A key finding in the current study is that JMJD2A promotes cardiac hypertrophy under pathological conditions. This is supported by both gain-of- and loss-of-function studies presented here. While overexpression of JMJD2A in mice exacerbates the hypertrophic response to pressure overload, inactivation of Jmjd2a blunts it (Figures and , respectively). Since JMJD2A is upregulated in human HCM patients (Figure ), we speculate that this upregulation of JMJD2A may play an active role in promoting cardiac hypertrophy in humans under cardiac stress conditions. It is noted that the mechanism by which the JMJD2A activity is regulated in mice may be different from that in humans, since we did not observe upregulation of JMJD2A expression in TAC-induced hypertrophy in our mouse model (data not shown). Rather, we found enhanced binding of JMJD2A to the promoter of prohypertrophic genes such as FHL1 in response to TAC.
Our studies suggest that JMJD2A may promote cardiac hypertrophy through FHL1. This is supported by the following evidence: FHL1 is known to mediate pressure overload–induced cardiac hypertrophy (25
), and overexpression of FHL1 in cardiomyocyte upregulates fetal gene expression (Supplemental Figure 4). JMJD2A activates the transcription of FHL1 both in vitro and in vivo and in response to hypertrophic stimuli (Figures –). The phenotype of cardiac inactivation of Jmjd2a
is consistent with that of FHL1
KO mice (Figure ). The MAPK-signaling pathway regulated by FHL1 is also affected by JMJD2A (Figure ). It remains to be determined whether FHL1 is the major downstream effector of JMJD2A. This could be determined by examining the phenotype of Jmjd2a
-Tg and FHL1
-null compound mice and testing to determine whether inactivation of FHL1
blunts the TAC-induced hypertrophy in Jmjd2a
-Tg mice. It is likely that JMJD2A may have other transcriptional targets during hypertrophic remodeling. We have shown that JMJD2A can synergistically activate ANP and sm22 transcription with SRF/myocardin in vitro (Figure ). Myocardin/SRF has been shown to be involved in cardiac development and hypertrophic remodeling (32
). Whether these and/or other SRF/myocardin-targeted genes mediate the prohypertrophic function of JMJD2A in vivo remains to be determined in the future. A genome-wide ChIP followed by deep sequencing (ChIP-seq) with chromatins from Jmjd2a
-Tg and Jmjd2a
hKO hearts would provide means to identify further potential JMJD2A targets involved in cardiac hypertrophy and heart failure.
It is well established that histone modifications play important roles in gene transcription. Over the past decades, a great deal has been learned regarding histone acetylation and its role in cardiac remodeling (6
). By comparison, little is known about the function of histone methylation even though it is the most abundant form of histone modifications. Unlike histone acetylation, which is usually associated with gene activation, histone methylation can lead to either activation or repression of gene transcription, depending on the lysine residues, the degree of methylation status (mono-, di-, or trimethylation), and chromatin location. It was shown previously that JMJD1A, a di/monomethyl demethylase, can activate the expression of contractile genes in smooth muscle cells (34
). Our studies indicate that trimethyl demethylation of H3K9me3 at the FHL1
promoter by JMJD2A in cardiomyocytes promotes gene transcription. As JMJD2A upregulates other SRF-regulated genes such as ANP and sm22, it is tempting to speculate that demethylation of H3K9 on promoters of SRF-targeted genes activates gene transcription.
JMJD2A could activate the transcription of SRF-targeted genes by either providing binding sites that stabilize/increase the affinity for transcription factors or preventing binding of H3K9me3 to heterochromatin binding protein HP-1. Binding of HP-1 to H3K9me3 was known to mediate conversion of euchromatin to heterochromatin, resulting in silencing of gene transcription. Our studies favor the former possibility as KD of JMJD2A did not abolish the ability of SRF/myocardin to activate FHL1 transcription (Supplemental Figure 6), suggesting that FHL1 was not inactivated in the absence of JMJD2A. Furthermore, we observed an increased amount of SRF/myocardin binding to the FHL1 promoter that is associated with upregulation of JMJD2A and decreased levels of H3K9me3. Taken together, our data suggest a feed-forward mechanism for the synergy between SRF/myocardin and JMDJ2A; SRF/myocardin recruits JMJD2A to the FHL1 promoter. In turn, JMJD2A demethylates H3K9me3, generating a surface/binding area for further recruitment of SRF/myocardin.
JMJD2A is a global regulator of chromatin remodeling and gene expression, and yet deletion of JMJD2A affects the transcription of only a handful of genes. While this finding may seem paradoxical, it is not unexpected. Gene expression is regulated through the action of transcription factors and histone-modifying enzymes. Many different histone-modifying enzymes, including HDACs, HATs, HMTs, and HDMs, contribute to the dynamic regulation of chromatin structure and function, with concomitant impacts on gene transcription. Unlike transcription factors that often have on-off effects on gene transcription, the effects of histone-modifying enzymes on gene transcription are often modulatory. This modulatory effect can be context- and gene-dependent such that only those genes exceeding the threshold will yield a phenotype and be identified. Therefore, it is not surprising that deletion of JMJD2A in the heart resulted in changes of the transcription levels of only a few target genes. However, it is worth noting that even though the number of genes whose expressions are affected by deletion of Jmjd2a is small, the genome-wide H3K9me3 marks affected by Jmjd2a deficiency may still be large. It will be interesting to identify these marks using ChIP-seq and to further investigate the relationship between JMJD2A-regulated H3K9me3 marks and other chromatin marks, i.e., histone code, for such a relationship may ultimately determine the transcriptional state of the gene as either active, repressed, or poised for activation.
In summary, our studies indicate that JMJD2A promotes cardiac hypertrophy in response to hypertrophic stimuli. JMJD2A demethylates H3K9me3 and activates transcription of prohypertrophic genes synergistically with SRF/myocardin. It is noted that the effect of JMJD2A on the expression of SRF/myocardin-targeted genes is modulatory, as loss of function of Jmjd2a does not abolish the ability of SRF/myocardin to activate gene transcription. JMJD2A could be a potential drug target for transcriptional therapy against cardiac hypertrophy and heart failure. It will be worth determining in the future whether small molecules designed to target the demethylase activity of JMJD2A could reduce and normalize the expression of SRF/myocardin-targeted genes that are upregulated during cardiac remodeling without compromising the physiological cardiac growth and functions of these genes that are part of the adaptive response to altered conditions.