Tagged actin mRNA dysregulation in IGF2BP1[Formula: see text] mice.

Gene expression is tightly regulated by RNA-binding proteins (RBPs) to facilitate cell survival, differentiation, and migration. Previous reports have shown the importance of the Insulin-like Growth Factor II mRNA-Binding Protein (IGF2BP1/IMP1/ZBP1) in regulating RNA fate, including localization, transport, and translation. Here, we generated and characterized a knockout mouse to study RBP regulation. We report that IGF2BP1 is essential for proper brain development and neonatal survival. Specifically, these mice display disorganization in the developing neocortex, and further investigation revealed a loss of cortical marginal cell density at E17.5. We also investigated migratory cell populations in the IGF2BP1[Formula: see text] mice, using BrdU labeling, and detected fewer mitotically active cells in the cortical plate. Since RNA localization is important for cellular migration and directionality, we investigated the regulation of ß-actin messenger RNA (mRNA), a well-characterized target with established roles in cell motility and development. To aid in our understanding of RBP and target mRNA regulation, we generated mice with endogenously labeled ß-actin mRNA (IGF2BP1[Formula: see text]; ß-actin-MS2[Formula: see text]). Using endogenously labeled ß-actin transcripts, we report IGF2BP1[Formula: see text] neurons have increased transcription rates and total ß-actin protein content. In addition, we found decreased transport and anchoring in knockout neurons. Overall, we present an important model for understanding RBP regulation of target mRNA.

Transcriptional burst fraction and size dynamics during lens fiber cell differentiation and detailed insights into the denucleation process.

Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell differentiation. In the ocular lens, the positions of lens fiber cells correlate precisely with their differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the ß-actin and lens differentiation-specific α-, ß-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular differentiation dramatically alters the burst fraction in synchronized waves across the lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.

Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site.

Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.

A transgenic mouse for in vivo detection of endogenous labeled mRNA.

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential ß-actin gene. As ß-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the ß-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single ß-actin mRNA molecules in various mouse tissues.

Bidirectional Analysis of Cryba4-Crybb1 Nascent Transcription and Nuclear Accumulation of Crybb3 mRNAs in Lens Fibers.

Purpose: Crystallin gene expression during lens fiber cell differentiation is tightly spatially and temporally regulated. A significant fraction of mammalian genes is transcribed from adjacent promoters in opposite directions ("bidirectional" promoters). It is not known whether two proximal genes located on the same allele are simultaneously transcribed. Methods: Mouse lens transcriptome was analyzed for paired genes whose transcriptional start sites are separated by less than 5 kbp to identify coexpressed bidirectional promoter gene pairs. To probe these transcriptional mechanisms, nascent transcription of Cryba4, Crybb1, and Crybb3 genes from gene-rich part of chromosome 5 was visualized by RNA fluorescent in situ hybridizations (RNA FISH) in individual lens fiber cell nuclei. Results: Genome-wide lens transcriptome analysis by RNA-seq revealed that the Cryba4-Crybb1 pair has the highest Pearson correlation coefficient between their steady-state mRNA levels. Analysis of Cryba4 and Crybb1 nascent transcription revealed frequent simultaneous expression of both genes from the same allele. Nascent Crybb3 transcript visualization in "early" but not "late" differentiating lens fibers show nuclear accumulation of the spliced Crybb3 transcripts that was not affected in abnormal lens fiber cell nuclei depleted of chromatin remodeling enzyme Snf2h (Smarca5). Conclusions: The current study shows for the first time that two highly expressed lens crystallin genes, Cryba4 and Crybb1, can be simultaneously transcribed from adjacent bidirectional promoters and do not show nuclear accumulation. In contrast, spliced Crybb3 mRNAs transiently accumulate in early lens fiber cell nuclei. The gene pairs coexpressed during lens development showed significant enrichment in human "cataract" phenotype.

Visualization of dynamics of single endogenous mRNA labeled in live mouse.

The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all ß-actin mRNA is fluorescently labeled. We found that ß-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple ß-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of ß-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

Variegated gene expression caused by cell-specific long-range DNA interactions.

Mammalian genomes contain numerous regulatory DNA sites with unknown target genes. We used mice with an extra ß-globin locus control region (LCR) to investigate how a regulator searches the genome for target genes. We find that the LCR samples a restricted nuclear subvolume, wherein it preferentially contacts genes controlled by shared transcription factors. No contacted gene is detectably upregulated except for endogenous ß-globin genes located on another chromosome. This demonstrates genetically that mammalian trans activation is possible, but suggests that it will be rare. Trans activation occurs not pan-cellularly, but in 'jackpot' cells enriched for the interchromosomal interaction. Therefore, cell-specific long-range DNA contacts can cause variegated expression.

Coenzyme Q10 and lipid-related gene induction in HeLa cells.

OBJECTIVE: Coenzyme Q10 is an antioxidant that may have a therapeutic role in cervical cancer. STUDY DESIGN: We investigated the cellular and molecular effects of 30 micromol/L Coenzyme Q10 in HeLa cells. Cell growth assays, fluorescence-activated cell sorting analyses, and Oil Red O staining were performed. Microarray experiments were performed in duplicate and analyzed on the basis of 2-fold changes in levels of gene expression. RESULTS: Coenzyme Q10 inhibited cell growth and led to apoptosis. Microarray analysis showed that 264 sequences were altered over time, with enrichment in lipid-related genes. Enhanced lipid accumulation was confirmed with Oil Red O staining. CONCLUSION: A lipid response to Coenzyme Q10 may affect mechanisms of growth inhibition in HeLa cells.

Tumor necrosis factor-alpha related gene response to Epothilone B in ovarian cancer.

OBJECTIVES: Epothilone B (EpoB) is a non-taxane microtubule-stabilizing agent with a mode of action similar to that of paclitaxel, but with the advantage of being active in paclitaxel-resistant cells. Knowledge regarding other mechanisms of EpoB action is limited. The purpose of this study was to identify gene expression profiles associated with the biological response to EpoB in an ovarian cancer cell line (SKOV3). METHODS: SKOV3 cells were maintained in McCoy's 5A media. Equal densities cells were treated with or without EpoB, and were evaluated for cell growth arrest and apoptosis. mRNA expression was evaluated by cDNA microarrays and quantitative, real time reverse transcription polymerase chain reaction (QRTPCR). RESULTS: EpoB (10 nM) led to cell cycle arrest and apoptosis in SKOV3 cells. Microarray analysis, comparing EpoB-treated to untreated cells, revealed altered expression of 41 genes. There was a predominance of sequences related to the TNFalpha stress response pathway. Differential expression of selected genes was confirmed by QRTPCR. CONCLUSIONS: We demonstrated that cDNA microarrays are a useful tool to rapidly screen for patterns of gene expression that characterize drug response. The microarray data suggest that the microtubule-stabilizing agent, EpoB, triggers stress-related signal transduction pathways related to TNFalpha. These pathways may contribute to mechanisms of EpoB action and potential mechanisms of resistance in ovarian cancer.