TRANSCRIPTIONAL REGULATION OF THE B1AR BY C-MYC

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们的实验室最近在？肾上腺素能受体？AR信号在胎儿和产后早期心肌细胞增殖调控中的作用。我们也在发展中的动物身上证明了？1AR的表达受一个新的序列元件控制，包括同源结构域蛋白结合位点、bHLH因子myc和max结合的E-box和糖皮质激素受体结合的共识位点。使用核连续试验来测量转录率，我们发现有一个更高的？在心脏生长“增殖期”的ar转录率高于老年动物这个增长？1AR转录率与原癌基因c-myc转录率的增加相关。在基因工程过表达c-myc （myc+/+）的大鼠成纤维细胞系中进行的转录率测定显示？1AR表达，与野生型细胞系比较。瞬时转染？1AR启动子构建体在myc+/+细胞中表现出强烈的表达，这种表达可以通过myc/max结合位点的突变或与c-myc反义表达载体的共转染而消除。虽然bHLH转录因子c-myc具有经典的DNA结合基序，但其作为转录因子的直接证明更为困难。我们最近的研究结果表明，心脏？1AR的转录和c-myc的表达是紧密结合的。在这个建议中，我们试图通过一个被广泛接受的实验生长模式来验证这种调节的生理重要性，即分离的心肌细胞的体外拉伸。我们还试图确定负责发育调节的1AR表达的转录机制。下面列出了最初的具体目标以及每个目标的进展情况。具体目标1：确定拉伸对？使用启动子报告基因构建，Northern blot和转录率测定1AR和c-myc。a.进展：如前所述，已从2-3天大的大鼠中分离出原代心肌细胞1,2细胞在专门设计的胶原基质上培养，用于屈肌细胞装置。经过24小时的夜间培养和稳定，细胞被拉伸的时间间隔从30分钟到8小时不等。先前的研究观察拉伸对c-myc激活的影响，表明原癌基因c-myc在拉伸两小时后达到峰值表达。然后从拉伸或控制的细胞中分离总RNA并进行Northern blot分析。Northern印迹用随机标记的带有c-myc或？1AR的cdna进行检测。结果如图1所示。在图A中可以看到拉伸两小时对c-myc原癌基因表达的影响。可以看到，经过两个小时的噬菌体，c-myc的表达显著增加。然而，在这一早期时间点，c-myc表达并未随之增加。拉伸0、2、4和8小时对？然后检测1AR mRNA。有惊人的（5-10倍）增长？1基于“增大化现实”技术的表达式。c-myc表达增加的时间顺序随后是？1AR的表达支持我们的初步数据，表明？1AR是myc的下游靶点。这将在这些细胞中进一步探讨，使用瞬时转染测定法在拉伸之前消除c-myc表达。我们假设？在用c-myc反义表达载体共转染后c-myc表达减弱的细胞中不会出现1AR如果转染效率不足以证明这种效果，我们将探索两种替代策略。其中之一是试图用RNA干扰来消除c-myc的表达。然而，这将依赖于类似的转染效率的变化。或者，我们可以构建含有反义方向c-myc编码序列的腺病毒表达载体。我们在瞬时转染试验和荧光素酶报告中已经成功地使用了这种策略。腺病毒介导转染的优点是可以成功感染相当大比例的分离心肌细胞，并检查取消c-myc表达对心肌细胞的影响。1AR转录率。特异性目标2：确定c-myc作为DNA结合蛋白和转录激活因子的作用？通过染色质免疫沉淀（ChIP）检测1AR基因。a.进展：正如最初申请中概述的那样，新的转录元件调节？1肾上腺素能受体表达。我们称之为糖皮质激素调节单元（GRU）的复合元件由同源结构域蛋白结合区域、myc和max结合的E-box和糖皮质激素反应元件组成。我们最近已经成功地鉴定了假定的同源结构域蛋白与上游区域的结合。用同源域和E-box结合区的寡核苷酸连接物进行亲和层析。分离细胞的萃取物首先通过肝素柱清洗。用凝胶移位法监测洗脱液。然后将适当的组分应用于DNA亲和柱上，并使用0.1-1 mM不等的KCl浓度逐步洗脱。柱洗脱液再次使用凝胶移位测定法进行监测。在0.3 - 0.4 mm的KCL馏分中鉴定出一个特定的条带，然后将其汇集，使用Amicon过滤器浓缩并进行SDS页电泳。在SDS页面凝胶上可以看到一些离散的条带。为了鉴定具有DNA结合活性的特定条带，将SDS page blot转移到尼龙膜上，并用含有同源结构域和E-box区域的放射性标记的寡核苷酸序列进行探针，即所谓的“西南印迹”。西南印迹法鉴定的特异条带从凝胶中洗脱，并在马萨诸塞州伍斯特大学进行质谱分析（MALDI-tof）。所鉴定的蛋白是先前已知的转录因子，称为多嘧啶束结合蛋白剪接因子（PTB-psf ref 3）。该转录因子对胰岛素样生长因子1 （IGF1）启动子的调控被广泛研究。它最初是由Randall Urban博士（UT，加尔维斯顿）分离出来的。Urban博士慷慨地提供了PTB-psf表达载体以及PTB-psf抗体。我们已经将GRU荧光素酶报告基因结构与PTB-psf单独或与c-myc联合表达。研究是在分离的3天大的原代心肌细胞中进行的。可以看出，单独的PTB-psf对gru依赖性荧光素酶活性只有适度的影响。然而，PTB-psf和c-myc的结合导致GRU荧光素酶结构的表达显著（5-6倍）增加。b.未来研究：正在进行的研究使用定点诱变去除同源结构域结合区，并证明PTB-psf的作用确实依赖于完整的转录元件。我们正在计划使用染色质免疫沉淀法进行实验，以确定myc和PTB-psf在？ar和其他发起人可以合作的地方。同样，我们正在进行单次和双次免疫沉淀试验，看看我们是否可以证明PTB-psf和c-myc之间的蛋白-蛋白相互作用。最后，我们认为？1AR在不同发育阶段的转录依赖于PTB-psf、c-myc、max、mad和糖皮质激素受体的化学计量学。RNAse保护试验和Western免疫印迹法正在进行，以确定这些重要的ar调节因子在RNA和蛋白质水平上的表达水平的个体发生。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our lab has recently demonstrated an important role for ? adrenergic receptor (?AR) signaling in regulation of cardiomyocyte proliferation during the fetal and early postnatal period. We have also shown in developing animals that ?1AR expression is under control of a novel sequence element(s) including a homeodomain protein binding site, an E-box for binding of the bHLH factors myc and max and a consensus site for glucocorticoid receptor binding. Using nuclear run-on assays for measurement of transcription rate, we showed that there is a much higher ?1AR transcription rate during the ¿proliferative period¿ of cardiac growth than in older animals.1 This increase in ?1AR transcription rate is associated with a concomitant increase in transcription rate of the protooncogene c-myc. Transcription rate assays performed in rat fibroblast cell lines genetically engineered to over-express c-myc (myc+/+) show increased ?1AR expression, compared to the wild type cell line. Transient transfection of ?1AR promoter constructs in the myc+/+ cells demonstrated robust expression, which is abrogated by mutation of the myc/max binding site or by co-transfection with a c-myc antisense expression vector. While the bHLH transcription factor c-myc has classic DNA binding motifis, its direct demonstration as a transcription factor has proven more difficult. Our recent results suggest that the regulation of cardiac ?1AR transcription and expression of c-myc are tightly integrated. In this proposal we have sought to verify the physiological importance of this regulation using a well accepted experimental growth paradigm, in-vitro stretch of isolated cardiomyocytes. We also seek to identify the transcriptional mechanism(s) responsible for the developmentally regulated expression of the ?1AR. The original Specific Aims are listed below along with progress in each Aim. Specific Aim 1: Determine the effect of stretch on transcriptional activation of ?1AR and c-myc using promoter reporter gene constructs, Northern blot and transcription rate assays. a. Progress: Primary cardiomyocytes have been isolated from 2-3 day old rats as previously described.1,2 Cells were cultured on specially-designed collagen matrix for use with the flexor cell apparatus. After 24 hours overnight culturing and stabilization, the cells were stretched for time intervals ranging from 30 minutes to eight hours. Prior studies looking at the effect of stretch on c-myc activation indicated that peak expression of the protooncogene c-myc was seen after two hours of stretch. Total RNA was then isolated from the stretched or controlled cells and subjected to Northern blot analysis. The Northern blots are probed with random primary-labeled cDNAs for with c-myc or the ?1AR. The results are shown in Figure 1. In Panel A can be seen the effects of stretch for two hours on expression of the c-myc proto-oncogene. As can be seen, c-myc expression increased substantially by a two-hour phage. There was, however, no concomitant increase in c-myc expression at this early timepoint. te effects of stretch for 0, 2, 4 & 8 hours on ?1AR mRNA was then examined. There was a striking (5-10 fold) increase in ?1AR expression. The temporal sequence of increased c-myc expression followed by an increase in ?1AR expression is supportive of our preliminary data suggesting the ?1AR is downstream target of myc. This will be explored further in these cells using transient transfection assays to abrogate c-myc expression prior to stretching. We hypothesize that a downstream increase in transcription rate of the ?1AR will not be seen in cells in which the c-myc expression has been attenuated by co-transfection with a c-myc antisense expression vector.1 If the transfection efficiency is not sufficient to demonstrate such an effect, there are two alternative strategies we will explore. One of these is to attempt to abrogate c-myc expression using RNA interference. This, however, will be dependent on similar vagaries of transfection efficiency. Alternatively, we may construct an adenovirus expression vector containing the c-myc coding sequence in antisense orientation. We have had success with this strategy in transient transfection assays and luciferase reporters. The advantages of the adenovirus-mediated transfections will be to allow successful ¿infection¿ of a substantial proportion of the isolated cardiomyocytes and examination of the effect of abrogation of c-myc expression on ?1AR transcription rate. Specific Aim 2: Determine the role of c-myc as a DNA binding protein and transcriptional activator of the ?1AR gene by chromatin immunoprecipitation (ChIP) assays. a. Progress: As outlined in the original application, novel transcriptional elements regulate ?1 adrenergic receptor expression. A composite element we called glucocorticoid regulatory unit (GRU) is composed of a region for binding of homeodomain proteins, the E-box where myc and max bind and the glucocorticoid response element. We have recently had success identifying the putative homeodomain protein binding to the upstream region. Affinity chromatography was carried out with an oligonucleotide concatener of the homeodomain and the E-box binding region. Extract from isolated cells was first cleaned up by passage over a heparan column. The eluate was monitored using gel shift assays. The appropriate fractions were then applied to the DNA affinity column and eluted in stepwise fashion using a KCl concentrations varying from 0.1-1 mM. The column eluate was again monitored using the gel shift assays. A specific band was identified in the .3-.4 mm KCL fraction which was then pooled, concentrated using an Amicon filter and subjected to SDS page electrophoresis. A number of discrete bands were seen on the SDS page gel. In order to identify the specific band with DNA binding activity, the SDS page blot was transferred to a nylon membrane and probed with the radiolabelled oliqonucleotide sequence containing the homeodomain and the E-box region, a so-called ¿Southwestern blot.¿ The specific band identified by Southwestern blotting was eluted from the gel and subjected to analysis by mass spectrometry (MALDI-tof) at University of Massachusetts in Worcester. The identified protein is a previously recognized transcription factor known as the polypyrimidine tract binding protein splicing factor, PTB-psf ref 3. This transcription factor has been most extensively studied in its regulation of the insulin-like growth factor 1 (IGF1) promoter. It was originally isolated by Dr. Randall Urban (UT, Galveston). Dr. Urban has graciously provided a PTB-psf expression vector as well as antibodies to PTB-psf. We have co-expressed the GRU luciferase reporter construct with PTB-psf alone or in combination with c-myc. Studies were carried out in isolated three-day old primary cardiomyocytes. As can be seen, PTB-psf alone had only a modest effect on GRU-dependent luciferase activity. However, the combination of PTB-psf and c-myc resulted in a significant (5-6 fold) increase in the expression of the GRU luciferase construct. b. Future Studies: Studies are in progress using site-directed mutagenesis to abrogate the homeodomain binding region and to demonstrate that the effects of PTB-psf are indeed dependent on the intact transcriptional element. We are planning experiments using chromatin immunoprecipitation as outlined in the original proposal to identify the binding regions for myc and PTB-psf on the ?1AR and other promoters where they may cooperate. Likewise, we are carrying out single and double immunoprecipitation assays to see if we can demonstrate protein-protein interaction between PTB-psf and c-myc. Lastly, we believe that the level of ?1AR transcription at different stages of development dependent on the stoichiometry of the proteins PTB-psf, c-myc, max, mad and the glucocorticoid receptor. RNAse protection assays and Western immunoblotting are being conducted to determine the ontogeny of the levels of expression at the RNA and protein level of these important regulators of the ?1AR.