The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectoderm-derived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.

Metazoan transcriptional repressors regulate chromatin through diverse histone modifications. Contributions of individual factors to the chromatin landscape in development is difficult to establish, as global surveys reflect multiple changes in regulators. Therefore, we studied the conserved Hairy/Enhancer of Split family repressor Hairy, analyzing histone marks and gene expression in Drosophila embryos. This long-range repressor mediates histone acetylation and methylation in large blocks, with highly context-specific effects on target genes. Most strikingly, Hairy exhibits biochemical activity on many loci that are uncoupled to changes in gene expression. Rather than representing inert binding sites, as suggested for many eukaryotic factors, many regions are targeted errantly by Hairy to modify the chromatin landscape. Our findings emphasize that identification of active cis-regulatory elements must extend beyond the survey of prototypical chromatin marks. We speculate that this errant activity may provide a path for creation of new regulatory elements, facilitating the evolution of novel transcriptional circuits.

Activation of gene transcription in mammalian cells requires several classes of coactivators that participate in different steps of the activation cascade. Using conventional and affinity chromatography, we have isolated a human coactivator complex that interacts directly with the C-terminal domain (CTD) of RNA polymerase II (Pol II). The CTD-binding complex is structurally and functionally indistinguishable from our previously isolated CRSP coactivator complex. The closely related, but transcriptionally inactive, ARC-L complex failed to interact with the CTD, indicating a significant biochemical difference between CRSP and ARC-L that may, in part, explain their functional divergence. Electron microscopy and three-dimensional single-particle reconstruction reveals a conformation for CTD-CRSP that is structurally distinct from unliganded CRSP or CRSP bound to SREBP-1a, but highly similar to CRSP bound to the VP16 activator. Together, our findings suggest that the human CRSP coactivator functions, at least in part, by mediating activator-dependent recruitment of RNA Pol II via the CTD.

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

Transcription, the first step of gene expression, is exquisitely regulated in higher eukaryotes to ensure correct development and homeostasis. Traditional biochemical, genetic, and genomic approaches have proved successful at identifying factors, regulatory sequences, and potential pathways that modulate transcription. However, they typically only provide snapshots or population averages of the highly dynamic, stochastic biochemical processes involved in transcriptional regulation. Single-molecule live-cell imaging has, therefore, emerged as a complementary approach capable of circumventing these limitations. By observing sequences of molecular events in real time as they occur in their native context, imaging has the power to derive cause-and-effect relationships and quantitative kinetics to build predictive models of transcription. Ongoing progress in fluorescence imaging technology has brought new microscopes and labeling technologies that now make it possible to visualize and quantify the transcription process with single-molecule resolution in living cells and animals. Here we provide an overview of the evolution and current state of transcription imaging technologies. We discuss some of the important concepts they uncovered and present possible future developments that might solve long-standing questions in transcriptional regulation.

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington’s disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. Here, we report the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. We demonstrate that both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. Our results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus.

It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Here, we report evidence for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. Our studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions.

The establishment and maintenance of spermatogenesis in mammals requires specialized networks of gene expression programs in the testis. The gonad-specific TAF4b component of TFIID (formerly TAF(II)105) is a transcriptional regulator enriched in the mouse testis. Herein we show that TAF4b is required for maintenance of spermatogenesis in the mouse. While young Taf4b-null males are initially fertile, Taf4b-null males become infertile by 3 mo of age and eventually exhibit seminiferous tubules devoid of germ cells. At birth, testes of Taf4b-null males appear histologically normal; however, at post-natal day 3 gonocyte proliferation is impaired and expression of spermatogonial stem cell markers c-Ret, Plzf, and Stra8 is reduced. Together, these data indicate that TAF4b is required for the precise expression of gene products essential for germ cell proliferation and suggest that TAF4b may be required for the regulation of spermatogonial stem cell specification and proliferation that is obligatory for normal spermatogenic maintenance in the adult.

To study the role of the transcription factor Myc-interacting protein 1 (MIZ-1) in activating various target genes after induction with the microtubule disrupting agent T113242, we have used small interfering RNA duplexes (siRNAs) to knockdown the expression of MIZ-1. As expected, depletion of MIZ-1 resulted in the inhibition of T113242-dependent activation of the low-density lipoprotein receptor (LDLR) gene in hepatocytes. Cells transfected with MIZ-1 siRNAs also exhibited growth arrest. In addition, inhibition of the extracellular signal-regulated kinase (ERK) pathway inhibited T113242-induced nuclear accumulation of MIZ-1 and activation of LDLR. Gene expression microarray analysis under various induction conditions identified other T113242-activated genes affected by a decrease in MIZ-1 and inhibition of the ERK pathway. We also found that the accumulation of MIZ-1 in the nucleus is influenced by cell-cell contact and/or growth. Taken together, our studies suggest that MIZ-1 regulates a specific set of genes that includes LDLR and that the ERK pathway plays a role in the activation of target promoters by MIZ-1.