Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte.

Direct lineage conversion offers a new strategy for tissue regeneration and disease modelling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to the target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study this process using bulk genomic techniques. Here we used single-cell RNA sequencing to overcome this limitation and analysed global transcriptome changes at early stages during the reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs). Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells during reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed unexpected downregulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor, Ptbp1, revealed that it is a critical barrier for the acquisition of cardiomyocyte-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of new surface markers for the enrichment of iCMs. In summary, our single-cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in iCM induction.

Lin28a Regulates Pathological Cardiac Hypertrophic Growth Through Pck2-Mediated Enhancement of Anabolic Synthesis.

BACKGROUND: Hypertrophic response to pathological stimuli is a complex biological process that involves transcriptional and epigenetic regulation of the cardiac transcriptome. Although previous studies have implicated transcriptional factors and signaling molecules in pathological hypertrophy, the role of RNA-binding protein in this process has received little attention. METHODS: Here we used transverse aortic constriction and in vitro cardiac hypertrophy models to characterize the role of an evolutionary conserved RNA-binding protein Lin28a in pathological cardiac hypertrophy. Next-generation sequencing, RNA immunoprecipitation, and gene expression analyses were applied to identify the downstream targets of Lin28a. Epistatic analysis, metabolic assays, and flux analysis were further used to characterize the effects of Lin28a and its downstream mediator in cardiomyocyte hypertrophic growth and metabolic remodeling. RESULTS: Cardiac-specific deletion of Lin28a attenuated pressure overload-induced hypertrophic growth, cardiac dysfunction, and alterations in cardiac transcriptome. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 ( Pck2) mRNA and increased its transcript level. Increasing Pck2 was sufficient to promote hypertrophic growth similar to that caused by increasing Lin28a, whereas knocking down Pck2 attenuated norepinephrine-induced cardiac hypertrophy. Epistatic analysis demonstrated that Pck2 mediated, at least in part, the role of Lin28a in cardiac hypertrophic growth. Furthermore, metabolomic analyses highlighted the role for Lin28a and Pck2 in promoting cardiac biosynthesis required for cell growth. CONCLUSIONS: Our study demonstrates that Lin28a promotes pathological cardiac hypertrophy and glycolytic reprograming, at least in part, by binding to and stabilizing Pck2 mRNA.

Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

RATIONALE: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. OBJECTIVE: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. METHODS AND RESULTS: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. CONCLUSIONS: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.

Generation of an inducible fibroblast cell line for studying direct cardiac reprogramming.

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) through forced expression of cardiac-lineage specific transcription factors holds promise as an alternative strategy for cardiac regeneration. To facilitate research in iCM reprogramming, we generated a suite of new tools. We developed a transformed cell line derived from mouse embryonic fibroblasts (MEF). This fibroblast cell line (MEF-T) harbors an αMHC-eGFP reporter transgene for rapid detection of newly derived iCMs. The MEF-T cell line is highly proliferative and easily transfected and transduced, making it an ideal tool for transgene expression and genetic manipulation. Additionally, we generated a Tet-On inducible polycistronic iCM reprogramming construct for the temporal regulation of reprogramming factor expression. Furthermore, we introduced this construct into MEF-T and created an inducible reprogrammable fibroblast cell line. These tools will facilitate future research in cell fate reprogramming by enabling the temporal control of reprogramming factor expression as well as high-throughput screening using libraries of small molecules, noncoding RNAs, and siRNAs. genesis 54:398-406, 2016. © 2016 Wiley Periodicals, Inc.

Evolutionary etiology of high-grade astrocytomas.

Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.

Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway.

A drawback of gene therapy using adeno-associated virus (AAV) is the DNA packaging restriction of the viral capsid (<4.7 kb). Recent observations demonstrate oversized AAV genome transduction through an unknown mechanism. Herein, AAV production using an oversized reporter (6.2 kb) resulted in chloroform and DNase-resistant particles harboring distinct "fragment" AAV (fAAV) genomes (5.0, 2.4, and 1.6 kb). Fractionation experiments determined that only the larger "fragments" mediated transduction in vitro, and relatively efficient transduction was also demonstrated in the muscle, the eye, and the liver. In contrast with concatemerization-dependent large-gene delivery by split AAV, fAAV transduction is independent of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in vitro and in vivo while disproportionately reliant on the DNA strand-annealing protein Rad51C. Importantly, fAAV's unique dependence on DNA repair proteins, compared with intact AAV, strongly suggests that the majority of oversized AAV transduction is mediated by fragmented genomes. Although fAAV transduction is less efficient than intact AAV, it is enhanced fourfold in muscle and sevenfold in the retina compared with split AAV transduction. Furthermore, fAAV carrying codon-optimized therapeutic dysferlin cDNA in a 7.5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy.

Exploring the Relationships between Multilevel Built Environments and Commute Durations in Dual-Earner Households: Does Gender Matter?

The links between built environments (BE) and commute durations have been extensively studied. However, relatively few studies have considered the effects of BEs at different spatial levels within a unified framework, or identified the gendered relationships between BEs and commute durations. Using survey data from 3209 household couples in 97 Chinese cities, this study investigates the effects of neighborhood- and city-level BEs on commute durations and the potential differences in these effects between the male and female members of the same household couple. A multi-group generalized multilevel structural equation model is applied to reveal the gendered relationships between neighborhood- and city-level BEs and commute durations. The findings suggest that the BE variables at two levels have significant effects on the commute duration. The mediating roles that the traffic congestion, car ownership, and commuting modes play in linking these BEs and commute durations are confirmed. Both levels of the BE variables are more influential factors for males' commuting durations. These findings have policy implications for the design of gender-equal transportation systems.

The Effect of Travel-Chain Complexity on Public Transport Travel Intention: A Mixed-Selection Model.

With urban expansion and traffic environment improvement, travel chains continue to grow, and the combination of travel purposes and modes becomes more complex. The promotion of mobility as a service (MaaS) has positive effects on facilitating the public transport traffic environment. However, public transport service optimization requires an accurate understanding of the travel environment, selection preferences, demand prediction, and systematic dispatch. Our study focused on the relationship between the trip-chain complexity environment and travel intention, combining the Theory of Planned Behavior (TPB) with travelers' preferences to construct a bounded rationality theory. First, this study used K-means clustering to transform the characteristics of the travel trip chain into the complexity of the trip chain. Then, based on the partial least squares structural equation model (PLS-SEM) and the generalized ordered Logit model, a mixed-selection model was established. Finally, the travel intention of PLS-SEM was compared with the travel sharing rate of the generalized ordered Logit model to determine the trip-chain complexity effects for different public transport modes. The results showed that (1) the proposed model, which transformed travel-chain characteristics into travel-chain complexity using K-means clustering and adopted a bounded rationality perspective, had the best fit and was the most effective with comparison to the previous prediction approaches. (2) Compared with service quality, trip-chain complexity negatively affected the intention of using public transport in a wider range of indirect paths. Gender, vehicle ownership, and with children/without children had significant moderating effects on certain paths of the SEM. (3) The research results obtained by PLS-SEM indicated that when travelers were more willing to travel by subway, the subway travel sharing rate corresponding to the generalized ordered Logit model was only 21.25-43.49%. Similarly, the sharing rate of travel by bus was only 32-44% as travelers were more willing to travel by bus obtained from PLS-SEM. Therefore, it is necessary to combine the qualitative results of PLS-SEM with the quantitative results of generalized ordered Logit. Moreover, when service quality, preferences, and subjective norms were based on the mean value, with each increase in trip-chain complexity, the subway travel sharing rate was reduced by 3.89-8.30%, while the bus travel sharing rate was reduced by 4.63-6.03%.

Exploring the Relationship between Built Environment and Commuting Mode Choice: Longitudinal Evidence from China.

The literature has offered much evidence regarding associations between the built environment (BE) and commuting behavior. However, most prior studies are conducted based on cross-sectional samples from developed countries, and little is known about the longitudinal link between BE and commuting behavior. Based on two rounds of survey data from China, this study examines relationships of BE with commuting mode choice from both cross-sectional and longitudinal perspectives. The effects of life-cycle events are considered within a unified framework. Results of the longitudinal examination of BE and commuting mode shift largely support the cross-sectional analysis. Specifically, promoting more balanced land use and improving residential density are important for car use reductions and active travel initiatives. Meanwhile, more balanced land use improves the probability of commuting by motorcycle and electric bike, but reduces the probability of commuting by public transit. This study also highlights the remarkable role played by life-cycle events in affecting commuting mode shifts.

Down-regulation of Beclin1 promotes direct cardiac reprogramming.

Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)-dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor-mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/ß-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.

Optimization of self-complementary AAV vectors for liver-directed expression results in sustained correction of hemophilia B at low vector dose.

Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Exploring the Influence of Built Environment on Car Ownership and Use with a Spatial Multilevel Model: A Case Study of Changchun, China.

Although the impacts of built environment on car ownership and use have been extensively studied, limited evidence has been offered for the role of spatial effects in influencing the interaction between built environment and travel behavior. Ignoring the spatial effects may lead to misunderstanding the role of the built environment and providing inconsistent transportation policies. In response to this, we try to employ a two-step modeling approach to investigate the impacts of built environment on car ownership and use by combining multilevel Bayesian model and conditional autocorrelation (CAR) model to control for spatial autocorrelation. In the two-step model, the predicting car ownership status in the first-step model is used as a mediating variable in the second-step car use model. Taking Changchun as a case study, this paper identifies the presence of spatial effects in influencing the effects of built environment on car ownership and use. Meanwhile, the direct and cascading effects of built environment on car ownership and use are revealed. The results show that the spatial autocorrelation exists in influencing the interaction between built environment and car dependency. The results suggest that it is necessary for urban planners to pay attention to the spatial effects and make targeted policy according to local land use characteristics.

A Loss of Function Screen of Epigenetic Modifiers and Splicing Factors during Early Stage of Cardiac Reprogramming.

Direct reprogramming of cardiac fibroblasts (CFs) to induced cardiomyocytes (iCMs) is a newly emerged promising approach for cardiac regeneration, disease modeling, and drug discovery. However, its potential has been drastically limited due to the low reprogramming efficiency and largely unknown underlying molecular mechanisms. We have previously screened and identified epigenetic factors related to histone modification during iCM reprogramming. Here, we used shRNAs targeting an additional battery of epigenetic factors involved in chromatin remodeling and RNA splicing factors to further identify inhibitors and facilitators of direct cardiac reprogramming. Knockdown of RNA splicing factors Sf3a1 or Sf3b1 significantly reduced the percentage and total number of cardiac marker positive iCMs accompanied with generally repressed gene expression. Removal of another RNA splicing factor Zrsr2 promoted the acquisition of CM molecular features in CFs and mouse embryonic fibroblasts (MEFs) at both protein and mRNA levels. Moreover, a consistent increase of reprogramming efficiency was observed in CFs and MEFs treated with shRNAs targeting Bcor (component of BCOR complex superfamily) or Stag2 (component of cohesin complex). Our work thus reveals several additional epigenetic and splicing factors that are either inhibitory to or required for iCM reprogramming and highlights the importance of epigenetic regulation and RNA splicing process during cell fate conversion.

Diabetes Exacerbates Myocardial Ischemia/Reperfusion Injury by Down-Regulation of MicroRNA and Up-Regulation of O-GlcNAcylation.

Management for patients with diabetes experiencing myocardial infarction remains a challenge. Here the authors show that hyperglycemia- and hyperinsulinemia-induced microRNA-24 (miR-24) reduction and O-GlcNAcylation in the diabetic heart contribute to poor survival and increased infarct size in diabetic myocardial ischemia/reperfusion (I/R). In a mouse model of myocardial I/R, pharmacological or genetic overexpression of miR-24 in hearts significantly reduced myocardial infarct size. Experimental validation revealed that miR-24 targets multiple key proteins, including O-GlcNac transferase, ATG4A, and BIM, to coordinately protect the myocardium from I/R injury. These results establish miR-24 as a promising therapeutic candidate for diabetic I/R injury.

p19(ARF) is dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression in vivo.

Recent studies have shown the p19(ARF) tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19(ARF) expression, was required for the response. Here, we use a genetic approach to test whether p19(ARF) functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19(ARF) has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19(ARF) gene. Thus, although p19(ARF) is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.

Magnetic resonance angiography visualization of abnormal tumor vasculature in genetically engineered mice.

Previous research on the vasculature of tumor-bearing animals has focused upon the microvasculature. Magnetic resonance angiography (MRA) offers a noninvasive, complementary approach that provides information about larger vessels. Quantitative analysis of MRA images of spontaneous preclinical tumor models has not been previously reported. Eleven TgT121;p53+/- mice, which invariably develop choroid plexus carcinoma (CPC), and nine age-matched healthy controls were imaged using T1, T2, and a high-resolution three-dimensional time-of-flight MRA sequences at 3 T. Tumors and vessels were segmented to determine tumor volume and vascular attributes, including number of terminal branches, vessel count, and the average vessel radii of MRA-visible vessels within the tumor. Differences in the vasculature between tumor-bearing animals and healthy controls were analyzed statistically. Although the spatial resolution of MRA prohibits visualization of capillaries, a high density of intratumor blood vessels was visualized in CPC mice. A significant increase in terminal branch count and vessel count, but not average vessel radius, was observed in CPCs when compared with normal controls. Both terminal branch count and vessel count were highly correlated with tumor volume. This study represents the first MRA analysis of a spontaneous preclinical brain tumor model. Although the spatial resolution of MRA is less than histologic analysis, MRA-obtained vascular attributes provide useful information with full brain coverage. We show that consistent tumor vasculature properties can be determined by MRA. Such methods are critical for developing preclinical therapeutic testing and will help guide the development of human brain tumor analyses.

Somatic induction of Pten loss in a preclinical astrocytoma model reveals major roles in disease progression and avenues for target discovery and validation.

High-grade astrocytomas are invariably deadly and minimally responsive to therapy. Pten is frequently mutated in aggressive astrocytoma but not in low-grade astrocytoma. However, the Pten astrocytoma suppression mechanisms are unknown. Here we introduced conditional null alleles of Pten (Pten(loxp/loxp)) into a genetically engineered mouse astrocytoma model [TgG(deltaZ)T121] in which the pRb family proteins are inactivated specifically in astrocytes. Pten inactivation was induced by localized somatic retroviral (MSCV)-Cre delivery. Depletion of Pten function in adult astrocytoma cells alleviated the apoptosis evoked by pRb family protein inactivation and also induced tumor cell invasion. In primary astrocytes derived from TgG(deltaZ)T121; Pten(loxp/loxp) mice, Pten deficiency resulted in a marked increase in cell invasiveness that was suppressed by inhibitors of protein kinase C (PKC) or of PKC-zeta, specifically. Finally, focal induction of Pten deficiency in vivo promoted angiogenesis in affected brains. Thus, we show that Pten deficiency in pRb-deficient astrocytoma cells contributes to tumor progression via multiple mechanisms, including suppression of apoptosis, increased cell invasion, and angiogenesis, all of which are hallmarks of high-grade astrocytoma. These studies not only provide mechanistic insight into the role of Pten in astrocytoma suppression but also describe a valuable animal model for preclinical testing that is coupled with a primary cell-based system for target discovery and drug screening.

Comparative Gene Expression Analyses Reveal Distinct Molecular Signatures between Differentially Reprogrammed Cardiomyocytes.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) or directly reprogrammed from non-myocytes (induced cardiomyocytes [iCMs]) are promising sources for heart regeneration or disease modeling. However, the similarities and differences between iPSC-CMs and iCMs are still unknown. Here, we performed transcriptome analyses of beating iPSC-CMs and iCMs generated from cardiac fibroblasts (CFs) of the same origin. Although both iPSC-CMs and iCMs establish CM-like molecular features globally, iPSC-CMs exhibit a relatively hyperdynamic epigenetic status, whereas iCMs exhibit a maturation status that more closely resembles that of adult CMs. Based on gene expression of metabolic enzymes, iPSC-CMs primarily employ glycolysis, whereas iCMs utilize fatty acid oxidation as the main pathway. Importantly, iPSC-CMs and iCMs exhibit different cell-cycle status, alteration of which influenced their maturation. Therefore, our study provides a foundation for understanding the pros and cons of different reprogramming approaches.

p53 must be competent for transcriptional regulation to suppress tumor formation.

In vitro studies suggest that effective tumor suppression by p53 requires multiple domains to execute transcription-dependent and transcription-independent functions. We generated a mutant p53 allele in mice, p53(W25QL26S) (p53(QS)), containing an inactive transactivation domain to evaluate the importance of transactivation for p53-mediated tumor suppression. Recently, we discovered that the allele also contains a valine substitution for alanine at codon 135, which borders the DNA-binding domain. We found that p53(QSval135) bound to chromatin albeit less well than p53(QSala135), but both were equally deficient in transcriptional regulation, apoptosis induction in mouse embryo fibroblasts (MEFs), and suppression of tumor formation by E1A, Ha-Ras transformed MEFs. p53(QSval135) mice and p53-null mice exhibited identical tumor development kinetics and spectra in spontaneous and oncogene-initiated tumorigenicity assays, when tested in a homo- and heterozygous configuration. The p53(QSval135) allele did not have dominant negative functions and behaved as a null allele. Taken together, these data indicate that effective tumor suppression requires the transcriptional regulation function of p53, and they suggest that transactivation independent functions of p53 are unlikely to contribute significantly to tumor suppression in vivo.

ErbB2 is required for cardiomyocyte proliferation in murine neonatal hearts.

It has been long recognized that the mammalian heart loses its proliferative capacity soon after birth, yet, the molecular basis of this loss of cardiac proliferation postnatally is largely unknown. In this study, we found that cardiac ErbB2, a member of the epidermal growth factor receptor family, exhibits a rapid and dramatic decline in expression at the neonatal stage. We further demonstrate that conditional ablation of ErbB2 in the ventricular myocardium results in upregulation of negative cell cycle regulators and a significant reduction in cardiomyocyte proliferation during the narrow neonatal proliferative time window. Together, our data reveal a positive correlation between the expression levels of ErbB2 with neonatal cardiomyocyte proliferation and suggest that reduction in cardiac ErbB2 expression may contribute to the loss of postnatal cardiomyocyte proliferative capacity.