Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy Ϸ4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.cAMP ͉ cAMP-response element binding protein-binding protein ͉ DNA methylation T he concept of a transcription code that dictates gene expression by means of the concerted action of multiple promoter elements has served as a useful paradigm for understanding specificity in gene regulation. The ability of multiple transcription factors to recruit RNA polymerase II to the promoter by means of low affinity interactions with components of the transcriptional machinery has been documented extensively (1).By contrast with this model, other studies suggest that some activators per se are sufficient to mediate transcriptional responses to hormonal signals depending on the occupancy of relevant sites (1). Indeed, genome-wide studies comparing binding patterns of hepatic nuclear factors in the liver and endocrine pancreas indicate that selective occupancy may often explain how different genetic programs are activated in distinct cell types (2).The cAMP-response element binding protein (CREB) family of activators stimulates cellular gene expression after phosphorylation at a conserved serine (Ser-133 in CREB1) in response to cAMP (3). Ser-133 phosphorylation promotes target gene activation in part by means of recruitment of the coactivator paralogs CREB-binding protein (CBP)͞p300 (4). Recruitment of CBP by phospho-CREB (P-CREB) appears sufficient for induction of cellular genes in response to cAMP (5, 6); in vitro transcription studies indicate that P-CREB is capable of promoting assembly of the transcriptional apparatus independent of other regulatory inputs (7).By contrast, some reports suggest that other upstream activators in addition to CREB are required for cellular gene induction by cAMP (8). Indeed, the notion that CREB coordinates with other transcription factors is supported by recent animal studies in which CREB appeared to elicit the expressi...

Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79). In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES) cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation) at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

Mammalian SWI/SNF chromatin remodeling complexes are involved in critical aspects of cellular growth and genomic stability. Each complex contains one of two highly homologous ATPases, BRG1 and BRM, yet little is known about their specialized functions. We show that BRG1and BRM associate with different promoters during cellular proliferation and differentiation, and in response to specific signaling pathways by preferential interaction with certain classes of transcription factors. BRG1 binds to zinc finger proteins through a unique N-terminal domain that is not present in BRM. BRM interacts with two ankyrin repeat proteins that are critical components of Notch signal transduction. Thus, BRG1 and BRM complexes may direct distinct cellular processes by recruitment to specific promoters through protein-protein interactions that are unique to each ATPase.

Recruitment of modifiers and remodelers to specific DNA sites within chromatin plays a critical role in controlling gene expression. The study of globin gene regulation provides a convergence point within which to address these issues in the context of tissue-specific and developmentally regulated expression. In this regard, erythroid Krüppel-like factor (EKLF) is critical. EKLF is a red cell-specific activator whose presence is crucial for establishment of the correct chromatin structure and high-level transcriptional induction of adult ␤-globin. We now find, by metabolic labeling-immunoprecipitation experiments, that EKLF is acetylated in the erythroid cell. EKLF residues acetylated by CREB binding protein (CBP) in vitro map to Lys-288 in its transactivation domain and Lys-302 in its zinc finger domain. Although site-specific DNA binding by EKLF is unaffected by the acetylation status of either of these lysines, directed mutagenesis of Lys-288 (but not Lys-302) decreases the ability of EKLF to transactivate the ␤-globin promoter in vivo and renders it unable to be superactivated by coexpressed p300 or CBP. In addition, the acetyltransferase function of CBP or p300 is required for superactivation of wild-type EKLF. Finally, acetylated EKLF has a higher affinity for the SWI-SNF chromatin remodeling complex and is a more potent transcriptional activator of chromatin-assembled templates in vitro. These results demonstrate that the acetylation status of EKLF is critical for its optimal activity and suggest a mechanism by which EKLF acts as an integrator of remodeling and transcriptional components to alter chromatin structure and induce adult ␤-globin expression within the ␤-like globin cluster.Recent advances in reconstructing transcriptional regulatory events have relied on biochemical and genetic studies that identified the basal transcription machinery and its activators, along with functional studies that delineated how these molecules work together to activate transcription, both on naked DNA and on DNA packaged into chromatin. A major insight into this mechanism has been that the dynamic range of transcription is greatly accentuated by the use of chromatinized templates, which are fully repressed compared to naked DNA, and that optimal induction begins from this repressed state rather than from the basal (or ground) state observed on naked DNA (7,35,66,73). It is within this system that chromatin modifiers and remodelers play a critical role (36, 80). Chromatin modifiers acetylate (e.g., CREB binding protein [CBP], p300, P/CAF) or deacetylate (e.g., histone deacetylases) histones at specific lysines within their amino termini, resulting in altered DNA binding affinities and a looser or tighter chromatin structure (15,67,80). Chromatin remodelers are multiprotein complexes (e.g., SWI-SNF and NURF) that utilize the energy from ATP hydrolysis to reorganize chromatin to a more open and accessible structure and do not covalently modify histones in the process (68,70,74). Transcriptional activators or repressors m...

Phytochromes are informational photoreceptors through which plants adapt their growth and development to prevailing light conditions. These adaptations are effected primarily through phytochrome regulation of gene expression by mechanisms that remain unclear. We describe a new mutant, hfr1 (long hypocotyl in far-red), that exhibits a reduction in seedling responsiveness specifically to continuous far-red light (FRc), thereby suggesting a locus likely to be involved in phytochrome A (phyA) signal transduction. Using an insertionally tagged allele, we cloned the HFR1 gene and subsequently confirmed its identity with additional alleles derived from a directed genetic screen. HFR1 encodes a nuclear protein with strong similarity to the bHLH family of DNA-binding proteins but with an atypical basic region. In contrast to PIF3, a related bHLH protein previously shown to bind phyB, HFR1 did not bind either phyA or B. However, HFR1 did bind PIF3, suggesting heterodimerization, and both the HFR1/PIF3 complex and PIF3 homodimer bound preferentially to the Pfr form of both phytochromes. Thus, HFR1 may function to modulate phyA signaling via heterodimerization with PIF3. HFR1 mRNA is 30-fold more abundant in FRc than in continuous red light, suggesting a potential mechanistic basis for the specificity of HFR1 to phyA signaling. Plants modify their growth and development in ways that allow them to adapt to their immediate environment. They do so by sensing a variety of environmental parameters and integrating the resulting information into coherent developmental decisions. Light is crucial to a plant's survival, and thus it is not surprising that plants have evolved an intricate set of photoreceptor systems through which they can track this parameter (Kendrick and Kronenberg 1994;Fankhauser and Chory 1997). The regulatory photoreceptors that sense red light (R) and far-red light (FR) are the phytochromes. These molecules undergo photoconversion between two spectroscopically and conformationally distinct forms, Pr (Rabsorbing) and Pfr (FR-absorbing), and conversion to Pfr is required for signal transmission. We are interested in the mechanisms by which phytochrome photoconversion effects change in gene expression.Seedling de-etiolation is not only an important phytochrome-regulated phase of development but also provides a convenient model system for dissecting the molecular basis of phytochrome signal transduction. Deetiolation can be thought of as a switch between two developmental programs: from skotomorphogenesis (or etiolation) in darkness to photomorphogenesis in light (McNellis and Deng 1995). These two programs differ in aspects that range from macroscopic morphology to the expression of a large number of light-regulated genes (Terzaghi and Cashmore 1995).Though there are five phytochromes in Arabidopsis, designated phyA through phyE (Clack et al. 1994), two of these, phyA and phyB, predominate in the regulation of seedling de-etiolation (Reed et al. 1994). Most aspects of de-etiolation can be induced by either R or FR...

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Embryonic stem (ES) cells require a coordinated network of transcription factors to maintain pluripotency or trigger lineage specific differentiation. Central to these processes are the proteins Oct4, Nanog, and Sox2. Although the transcriptional targets of these factors have been extensively studied, very little is known about how the proteins themselves are regulated, especially at the post-translational level. Post-translational modifications are well documented to have broad effects on protein stability, activity, and cellular distribution. Here, we identify a key lysine residue in the nuclear export signal of Sox2 that is acetylated, and demonstrate that blocking acetylation at this site retains Sox2 in the nucleus and sustains expression of its target genes under hyperacetylation or differentiation conditions. Mimicking acetylation at this site promotes association of Sox2 with the nuclear export machinery. In addition, increased cellular acetylation leads to reduction in Sox2 levels by ubiquitination and proteasomal degradation, thus abrogating its ability to drive transcription of its target genes. Acetylation-mediated nuclear export may be a commonly used regulatory mechanism for many Sox family members, as this lysine is conserved across species and in orthologous proteins. STEM CELLS

Targeted, noninvasive neuromodulation of the brain of an otherwise awake subject could revolutionize both basic and clinical neuroscience. Toward this goal, we have developed nanoparticles that allow noninvasive uncaging of a neuromodulatory drug, in this case the small molecule anesthetic propofol, upon the application of focused ultrasound. These nanoparticles are composed of biodegradable and biocompatible constituents and are activated using sonication parameters that are readily achievable by current clinical transcranial focused ultrasound systems. These particles are potent enough that their activation can silence seizures in an acute rat seizure model. Notably, there is no evidence of brain parenchymal damage or blood-brain barrier opening with their use. Further development of these particles promises noninvasive, focal, and image-guided clinical neuromodulation along a variety of pharmacological axes.