Clifford L. Wang

Activation of an oncogenic microRNA cistron by provirus integration

Retroviruses can cause tumors when they integrate near a protooncogene or tumor suppressor gene of the host. We infected >2,500 mice with the SL3-3 murine leukemia virus; in 22 resulting tumors, we found provirus integrations nearby or within the gene that contains the mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR, we showed that expression of miRNA was increased in these tumors, indicating that retroviral infection can induce expression of oncogenic miRNAs. Our results demonstrate that retroviral mutagenesis can be a potent tool for miRNA discovery.

Expression of Aire and the Early Wave of Apoptosis in Spermatogenesis

Expression of the autoimmune regulator (Aire) protein in mice and humans is thought to be restricted to the medullary epithelial and monocyte-dendritic cells of the thymus. There it mediates expression and presentation of a large variety of proteins, including those that are peripheral organ-specific and are not expressed by other thymocytes. In this way, self-reactive T lymphocytes that would attack peripheral cells producing these proteins are confronted with the self-Ags and, as a consequence, are deleted. In this study, we show that Aire mRNA is also expressed in the testis—another tissue with promiscuous gene expression. Aire protein, however, is expressed only sporadically in spermatogonia and spermatocytes. Transcription of genes that are under Aire control in the thymus is unaffected by Aire in the testis. However, in mice with a disrupted Aire gene, the scheduled apoptotic wave of germ cells, which is necessary for normal mature spermatogenesis, is reduced, and sporadic apoptosis in adults is increased. Because Rag-1 deficiency does not abolish the effect, the adaptive immune system is not involved. We suggest that there is a link between the scheduled and sporadic apoptotic processes and propose that scheduled apoptosis provides a counterselection mechanism that keeps the germline stable.

Tuning gene expression with synthetic upstream open reading frames

We engineered short ORFs and used them to control the expression level of recombinant proteins. These short ORFs, encoding a two-amino acid peptide, were placed upstream of an ORF encoding a protein of interest. Insertion of these upstream ORFs (uORFs) resulted in suppression of protein expression. By varying the base sequence preceding the uORF, we sought to vary the translation initiation rate of the uORF and subsequently control the degree of this suppression. Using this strategy, we generated a library of RNA sequence elements that can specify protein expression over a broad range of levels. By also using multiple uORFs in series and non-AUG start codons, we were able to generate particularly low expression levels, allowing us to achieve expression levels spanning three orders of magnitude. Modeling supported a mechanism where uORFs shunt the flow of ribosomes away from the downstream protein-coding ORF. With a lower translation initiation rate at the uORF, more ribosomes “leak” past the uORF; consequently, more ribosomes are able to reach and translate the downstream ORF. We report expression control by engineering uORFs and translation initiation to be robust, predictable, and reproducible across all cell types tested. We propose control of translation initiation as a primary method of choice for tuning expression in mammalian systems.

Quantitative analysis of mammalian translation initiation sites by FACS-seq.

An approach combining fluorescence‐activated cell sorting and high‐throughput DNA sequencing (FACS‐seq) was employed to determine the efficiency of start codon recognition for all possible translation initiation sites (TIS) utilizing AUG start codons. Using FACS‐seq, we measured translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed by a single motif, were also important for modeling TIS efficiency. Our dataset combined with modeling allowed us to predict genome‐wide translation initiation efficiency for all mRNA transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to identify candidate driver mutations consistent with known tumor expression patterns. Finally, we implemented a quantitative leaky scanning model to predict alternative initiation sites that produce truncated protein isoforms and compared predictions with ribosome footprint profiling data. The comprehensive analysis of the TIS sequence space enables quantitative predictions of translation initiation based on genome sequence.

Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states

Significance Population heterogeneity can make the treatment of tumors more challenging. Whereas a therapeutic agent may be effective against one fraction of a population, it may be less effective against another fraction. Although heterogeneity can be genetic and attributed to mutations, there can also be nongenetic heterogeneity, where a clonal population can harbor distinct subpopulations. Here, we identified a single gene, p21, that was responsible for population heterogeneity in cell cycle activity and explain that this heterogeneity can arise from regulatory relationships of p21 with Cyclin-dependent kinase 2 (CDK2) and E3 ubiquitin ligases. We suggest that, instead of using CDK inhibitors (CKIs) in cancer therapy, CKIs themselves should be targeted. Given concurrently with chemotherapy agents, CKI inhibitors would reduce tumor heterogeneity and thus increase chemotherapy efficacy. Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states—quiescence and cell cycling—can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21–CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.

A cell-compatible conductive film from a carbon nanotube network adsorbed on poly-L-lysine.

Single-walled carbon nanotubes (SWNTs) have shown promise for use in organic electronic applications including thin film transistors, conducting electrodes, and biosensors. Additionally, previous studies found applications for SWNTs in bioelectronic devices, including drug delivery carriers and scaffolds for tissue engineering. There is a current need to rapidly process SWNTs from solution phase to substrates in order to produce device structures that are also biocompatible. Studies have shown the use of surfaces covalently functionalized with primary amines to selectively adsorb semiconducting SWNTs. Here we report the potential of substrates modified with physisorbed polymers as a rapid biomaterials-based approach for the formation of SWNT networks. We hypothesized that rapid surface modification could be accomplished by adsorption of poly-L-lysine (PLL), which is also frequently used in biological applications. We detail a rapid and facile method for depositing SWNTs onto various substrate materials using the amine-rich PLL. Dispersions of SWNTs of different chiralities suspended in N-methylpyrrolidinone (NMP) were spin coated onto various PLL-treated substrates. SWNT adsorption and alignment were characterized by atomic force microscopy (AFM) while electrical properties of the network were characterized by 2-terminal resistance measurements. Additionally, we investigated the relative chirality of the SWNT networks by micro-Raman spectroscopy. The SWNT surface density was strongly dependent upon the adsorbed concentration of PLL on the surface. SWNT adsorbed on PLL-treated substrates exhibited enhanced biocompatibility compared to SWNT networks fabricated using alternative methods such as drop casting. These results suggest that PLL films can promote formation of biocompatible SWNT networks for potential biomedical applications.

Evaluation of sgRNA Target Sites for CRISPR-Mediated Repression of TP53

The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.

p53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress

Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.

DNA Acrobats of the Ig Class Switch

Small resting B lymphocytes all start out producing IgM Abs. Upon encountering Ag, the cells become activated and make a switch from IgM to other Ig classes. This class switch serves to distribute a particular V region to different Ig C regions. Each C region mediates a specialized effector function, and so, through switching, an organism can guide its Abs to various sites. Creating the new H chain requires loop-out and deletion of DNA between switch regions. These DNA acrobatics require transcription of the switch regions, presumably so that necessary factors can gain access to the DNA. These requisite switching factors include activation-induced cytidine deaminase and components of general DNA repair, including base excision repair, mismatch repair, and double-strand break repair. Despite much recent progress, not all important factors have been discovered, especially those that may guide recombination to a particular subclass.