We have introduced point mutations into both alleles of NCOR1 and SMRT that specifically abolish their ability to activate HDAC3. Using this unique mouse model we show that the DADs of nuclear receptor corepressors NCOR1 and SMRT are required for HDAC3 enzyme activity in vivo
. This finding is of great importance as HDAC3 has been shown to play pivotal roles in transcriptional regulation1,6,7
, cell cycle progression11–13
, developmental events10
, and metabolism16,17
. Our data clearly show that NCOR1 or SMRT is required for nearly all HDAC3 enzyme activity in vivo
HDAC3 clearly functions as an epigenomic modifier in the liver16
, and indeed histone acetylation was increased in the NS-DADm liver at genomic locations where HDAC3 normally bind. This is most likely explained by the loss of HDAC3 catalytic activity. In addition, HDAC3 occupancy on the genome was significantly reduced in the NS-DADm, demonstrating the importance of the NCOR1 and SMRT DADs in recruiting HDAC3 to the genome, and providing a second mechanism for increased histone acetylation at site of endogenous HDAC3 recruitment in WT mice. Histone acetylation and deacetylation alters chromosome accessibility and affect functions of transcription factors (TFs) acting at the genome2
. Many inhibitors of the enzyme activity of class I HDACs are being developed to treat diseases including several types of cancer44,45
. Moreover, approximately 10% of currently prescribed drugs directly target TFs46
, including tamoxifen for breast cancer and bicalutamide for prostate cancer, which target nuclear receptors by regulating their interaction with HDAC347,48
. Therefore our findings towards understanding basic HDAC biology have important therapeutic implications.
The NS-DADm mice exhibited mild hepatic steatosis, and molecular analysis revealed reduced or absent HDAC3 binding and increased local histone acetylation at upregulated lipid metabolic genes. Therefore these effects are likely due to the absence of HDAC3-dependent histone deacetylation. Nevertheless, mice lacking HDAC3 in liver manifest much more severe hepatic steatosis10,16,17
along with disrupted cholesterol homeostasis10,17
, whereas NS-DADm mice exhibited no detectable alteration in hepatic cholesterol. Furthermore, while absence of cardiac HDAC3 causes lethality or diet-induced heart failure depending on when the HDAC3 is deleted17,39
, NS-DADm mice have normal hearts and are able to tolerate a high fat diet (data not shown). Thus, while our studies show for the first time that the nuclear receptor corepressors are required for the deacetylase activity of HDAC3 in vivo
, they also suggest a DAD-independent or deacetylase-independent role of HDAC3. In addition to their roles in adult tissues, germ line deletion of NCOR126
, or HDAC310,39
all cause embryonic lethality. The viability of the N-DADm mice demonstrated that the DAD is not required for the essential functions of NCOR134
during embryonic development. The viability of the S-DADm mice makes this point about SMRT, which has a variety of functions besides HDAC3 activation49–52
. The phenotype of the S-DADm mice will be further characterized. More remarkably, however, although HDAC3 enzyme activity is diminished to an almost undetectable level in embryos of NS-DADm, the mice are viable and live to adulthood. They also suggest that HDAC3 has a DAD-independent or deacetylase-independent function that is required for life.
The DAD-independent functions of HDAC3 are unlikely to be the regulation of the genomic recruitment of NCOR1 and SMRT, since the genomic occupancy of these corepressors was maintained in the livers of NS-DADm as well as in mice lacking hepatic HDAC3. Also, while it is possible that HDAC3 has nuclear receptor corepressor-independent ability to deacetylate non-histone substrates53–58
, this is unlikely because the substrate used in the HDAC3 assay is a short peptide rather than a full-length histone protein. One caveat is that, due to the relatively high background of the HDAC activity assay, it is also possible that there is a small amount of residual HDAC3 deacetylase activity that is not detectable over background but may contribute to the modest phenotype of NS-DADm mice relative to mice lacking HDAC3 protein. It should also be noted that the genome-wide localization of HDAC3 was reduced but not abrogated in NS-DADm mice, potentially due to the second region in NCOR1 and SMRT that interacts with HDAC3 but does not activate the enzyme33
. Nonetheless, the present study in NS-DADm mice demonstrates for the first time that corepressors NCOR1 and SMRT are required for endogenous HDAC3 activity. It also raises the possibility that the HDAC3 protein is critical for embryonic development as well as adult physiology through non-enzymatic mechanisms.