Cardiac-specific deletion of ménage-à-trois-1 (MAT1)

The Cdk7/cyclin H/ménage-à-trois 1 (MAT1) heterotrimer has proposed functions in transcription as the kinase component of basal transcription factor TFIIH and is activated in adult hearts by hypertrophic pathways. Using cardiac-specific Cre, we ablated MAT1 in myocardium. Despite reduced Cdk7 activity, MAT1-deficient hearts grew normally. However, fatal heart failure ensued at 6-8 weeks. By microarray profiling, quantitative RT-PCR, and Western blotting at 4 weeks, genes for energy metabolism were found to be suppressed selectively, including targets of peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1). Cardiac metabolic defects were substantiated in isolated perfused hearts and isolated mitochondria. In culture, deleting MAT1 with Cre disrupted PGC-1 function: PGC-1α failed to activate PGC-1-responsive promoters and nuclear receptors, GAL4-PGC-1α was functionally defective, and PGC-1β likewise was deficient. PGC-1 was shown to interact with MAT1 and Cdk7, in co-precipitation assays. Thus, we demonstrate an unforeseen essential role for MAT1 in operation of the PGC-1 family of co-activators. Keywords: conditional knockout in mice MAT1F/F mice (Korsisaari et al., 2002) were bred with mice expressing Cre recombinase under the control of the cardiomyocyte-specific α-myosin heavy chain (αMHC) promoter (Gaussin et al., 2002a) and back-bred to MAT1F/F mice to generate the cardiac-specific knockout (αMHC-Cre+/0; MAT1F/F; CKO; Fig. 1A, B). Control mice were αMHC-Cre+/0; MAT1F/+ littermates, differing by the presence of one wild-type MAT1 allele, and excluding Cre-mediated toxicity as a basis for phenotypic disparity. Cardiac RNA samples were analyze at 2 or 4 weeks of age (N = 3-5 for each condition tested.

Cdk7/细胞周期蛋白H（cyclin H）/三联体蛋白1（ménage-à-trois 1, MAT1）异源三聚体，作为基础转录因子TFIIH（basal transcription factor TFIIH）的激酶组分，被认为在转录过程中发挥功能，且在成年心脏中可通过肥厚通路被激活。本研究采用心脏特异性Cre重组酶系统，在心肌细胞中敲除MAT1基因。尽管Cdk7活性有所降低，但MAT1敲除的心脏发育正常。然而，小鼠在6~8周龄时会出现致命性心力衰竭。在4周龄时通过基因芯片表达谱分析、定量逆转录聚合酶链式反应（quantitative reverse transcription PCR, qRT-PCR）及蛋白质免疫印迹（Western blotting）检测，我们发现能量代谢相关基因呈现选择性表达抑制，其中包括过氧化物酶体增殖物激活受体γ辅激活因子1（peroxisome proliferator-activated receptor-gamma coactivator-1, PGC-1）的靶基因。离体灌流心脏实验与分离线粒体实验进一步验证了心脏代谢缺陷表型。在细胞培养实验中，通过Cre介导的MAT1敲除会破坏PGC-1的功能：PGC-1α无法激活受PGC-1调控的启动子与核受体，GAL4-PGC-1α融合蛋白功能异常，且PGC-1β的活性同样受损。免疫共沉淀（co-precipitation）实验证实，PGC-1可与MAT1及Cdk7发生相互作用。综上，本研究揭示了MAT1在过氧化物酶体增殖物激活受体γ辅激活因子1（PGC-1）家族辅激活因子运作中此前未被发现的关键作用。关键词：小鼠条件性基因敲除。本研究将MAT1F/F基因敲除小鼠（Korsisaari等，2002）与心肌细胞特异性α-肌球蛋白重链（α-myosin heavy chain, αMHC）启动子驱动Cre重组酶表达的小鼠（Gaussin等，2002a）交配，并回交至MAT1F/F小鼠，以构建心脏特异性敲除小鼠模型（αMHC-Cre+/0; MAT1F/F; 即CKO；见图1A、B）。对照组小鼠为αMHC-Cre+/0; MAT1F/+同窝小鼠，仅携带一个野生型MAT1等位基因，以此排除Cre重组酶介导的毒性对表型差异的影响。我们在小鼠2周龄与4周龄时采集心脏RNA样本（每组检测样本量为3~5只）。