A RSC/nucleosome complex determines chromatin architecture and facilitates activator binding.

How is chromatin architecture established and what role does it play in transcription? We show that the yeast regulatory locus UASg bears, in addition to binding sites for the activator Gal4, sites bound by the RSC complex. RSC positions a nucleosome, evidently partially unwound, in a structure that facilitates Gal4 binding to its sites. The complex comprises a barrier that imposes characteristic features of chromatin architecture. In the absence of RSC, ordinary nucleosomes encroach over the UASg and compete with Gal4 for binding. Taken with our previous work, the results show that both prior to and following induction, specific DNA-binding proteins are the predominant determinants of chromatin architecture at the GAL1/10 genes. RSC/nucleosome complexes are also found scattered around the yeast genome. Higher eukaryotic RSC lacks the specific DNA-binding determinants found on yeast RSC, and evidently Gal4 works in those organisms despite whatever obstacle broadly positioned nucleosomes present.

Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis.

Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-ß family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-ß family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-ß families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors.

Biochemical and Cellular Analysis Reveals Ligand Binding Specificities, a Molecular Basis for Ligand Recognition, and Membrane Association-dependent Activities of Cripto-1 and Cryptic.

Transforming growth factor ß (TGF-ß) pathways are key determinants of cell fate in animals. Their basic mechanism of action is simple. However, to produce cell-specific responses, TGF-ß pathways are heavily regulated by secondary factors, such as membrane-associated EGF-CFC family proteins. Cellular activities of EGF-CFC proteins have been described, but their molecular functions, including how the mammalian homologs Cripto-1 and Cryptic recognize and regulate TGF-ß family ligands, are less clear. Here we use purified human Cripto-1 and mouse Cryptic produced in mammalian cells to show that these two EGF-CFC homologs have distinct, highly specific ligand binding activities. Cripto-1 interacts with BMP-4 in addition to its known partner Nodal, whereas Cryptic interacts only with Activin B. These interactions depend on the integrity of the protein, as truncated or deglycosylated Cripto-1 lacked BMP-4 binding activity. Significantly, Cripto-1 and Cryptic blocked binding of their cognate ligands to type I and type II TGF-ß receptors, indicating that Cripto-1 and Cryptic contact ligands at their receptor interaction surfaces and, thus, that they could inhibit their ligands. Indeed, soluble Cripto-1 and Cryptic inhibited ligand signaling in various cell-based assays, including SMAD-mediated luciferase reporter gene expression, and differentiation of a multipotent stem cell line. But in agreement with previous work, the membrane bound form of Cripto-1 potentiated signaling, revealing a critical role of membrane association for its established cellular activity. Thus, our studies provide new insights into the mechanism of ligand recognition by this enigmatic family of membrane-anchored TGF-ß family signaling regulators and link membrane association with their signal potentiating activities.

Determinants of nucleosome positioning and their influence on plant gene expression.

Nucleosome positioning influences the access of transcription factors (TFs) to their binding sites and gene expression. Studies in plant, animal, and fungal models demonstrate similar nucleosome positioning patterns along genes and correlations between occupancy and expression. However, the relationships among nucleosome positioning, cis-regulatory element accessibility, and gene expression in plants remain undefined. Here we showed that plant nucleosome depletion occurs on specific 6-mer motifs and this sequence-specific nucleosome depletion is predictive of expression levels. Nucleosome-depleted regions in Arabidopsis thaliana tend to have higher G/C content, unlike yeast, and are centered on specific G/C-rich 6-mers, suggesting that intrinsic sequence properties, such as G/C content, cannot fully explain plant nucleosome positioning. These 6-mer motif sites showed higher DNase I hypersensitivity and are flanked by strongly phased nucleosomes, consistent with known TF binding sites. Intriguingly, this 6-mer-specific nucleosome depletion pattern occurs not only in promoter but also in genic regions and is significantly correlated with higher gene expression level, a phenomenon also found in rice but not in yeast. Among the 6-mer motifs enriched in genes responsive to treatment with the defense hormone jasmonate, there are no significant changes in nucleosome occupancy, suggesting that these sites are potentially preconditioned to enable rapid response without changing chromatin state significantly. Our study provides a global assessment of the joint contribution of nucleosome occupancy and motif sequences that are likely cis-elements to the control of gene expression in plants. Our findings pave the way for further understanding the impact of chromatin state on plant transcriptional regulatory circuits.

Chromatin Remodeler Recruitment during Macrophage Differentiation Facilitates Transcription Factor Binding to Enhancers in Mature Cells.

We show how enhancers of macrophage-specific genes are rendered accessible in differentiating macrophages to allow their induction in mature cells in response to an appropriate stimulus. Using a lentiviral knockdown approach in primary differentiating macrophages from mouse bone marrow, we demonstrate that enhancers of Il12b and Il1a are kept relatively lowly occupied by nucleosomes and accessible through recruitment of the nucleosome remodeler BAF/PBAF. Our results using an inducible cell line that expresses an estrogen receptor fusion of the macrophage-specific transcription factor PU.1 (PUER) show that BAF/PBAF recruitment to these enhancers is a consequence of translocation of PUER to the nucleus in the presence of tamoxifen, and we speculate that remodeler recruitment may be directly mediated by PU.1. In the absence of BAF/PBAF recruitment, nucleosome occupancy at the enhancer of Il12b (and to a lesser extent at Il1a) reaches high levels in bone marrow-derived macrophages (BMDMs), and the enhancers are not fully cleared of nucleosomes upon LPS induction, resulting in impaired gene expression. Analysis of Il12b expression in single cells suggests that recruitment of the remodeler is necessary for high levels of transcription from the same promoter, and we propose that remodelers function by increasing nucleosome turnover to facilitate transcription factor over nucleosome binding in a process we have termed "remodeler-assisted competition."

Activator control of nucleosome occupancy in activation and repression of transcription.

The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose) and repression (by glucose) of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the action of SWI/SNF recruited by the activator, and a slower one that does not-clarifies our understanding of the early events of gene activation, and in particular corrects earlier reports that SWI/SNF plays no role in GAL gene induction. Our finding that chromatin structure is irrelevant for repression as studied here-that is, repression sets in as efficiently whether or not promoter nucleosomes are allowed to reform-contradicts the widely held, but little tested, idea that nucleosomes are required for repression. These findings were made possible by our nucleosome occupancy assay. The assay, we believe, will prove useful in studying other outstanding issues in the field.

HSP90/70 chaperones are required for rapid nucleosome removal upon induction of the GAL genes of yeast.

Induction of transcription of the GAL genes of yeast by galactose is a multistep process: Galactose frees the activator Gal4 of its inhibitor, Gal80, allowing Gal4 to recruit proteins required to transcribe the GAL genes. Here, we show that deletion of components of either the HSP90 or the HSP70 chaperone machinery delays this induction. This delay remains when the galactose-signaling pathway is bypassed, and it cannot be explained by a chaperone requirement for DNA binding by Gal4. Removal of promoter-bound nucleosomes is delayed in a chaperone mutant, and our findings suggest an involvement of HSP90 and HSP70 in this early step in gene induction.

Responses of four yeast genes to changes in the transcriptional machinery are determined by their promoters.

Many yeast genes are distinguished by their specific requirements for different components of the transcriptional machinery. Here we examine four genes that fall into two classes as defined by their dependence on specific components of the transcriptional machinery. We describe a series of hybrid constructs, each of which bears activator binding sites that are associated with a promoter other than that with which they are usually affiliated. We examine expression of these reporters in strains bearing three modifications of the transcriptional machinery. Our results indicate that, in each of these cases, the promoter (and not the activator) determines which components of the transcriptional machinery are required. These and additional results, including those of others, clarify how disparate activators can work at many different promoters.

The Lineage-Specific Transcription Factor PU.1 Prevents Polycomb-Mediated Heterochromatin Formation at Macrophage-Specific Genes.

Lineage-specific transcription factors (TFs) are important determinants of cellular identity, but their exact mode of action has remained unclear. Here we show using a macrophage differentiation system that the lineage-specific TF PU.1 keeps macrophage-specific genes accessible during differentiation by preventing Polycomb repressive complex 2 (PRC2) binding to transcriptional regulatory elements. We demonstrate that the distal enhancer of a gene becomes bound by PRC2 as cells differentiate in the absence of PU.1 binding and that the gene is wrapped into heterochromatin, which is characterized by increased nucleosome occupancy and H3K27 trimethylation. This renders the gene inaccessible to the transcriptional machinery and prevents induction of the gene in response to an external signal in mature cells. In contrast, if PU.1 is bound at the transcriptional regulatory region of a gene during differentiation, PRC2 is not recruited, nucleosome occupancy is kept low, and the gene can be induced in mature macrophages. Similar results were obtained at the enhancers of other macrophage-specific genes that fail to bind PU.1 as an estrogen receptor fusion (PUER) in this system. These results show that one role of PU.1 is to exclude PRC2 and to prevent heterochromatin formation at macrophage-specific genes.

Nucleosomes are stably evicted from enhancers but not promoters upon induction of certain pro-inflammatory genes in mouse macrophages.

Chromatin is thought to act as a barrier for binding of cis-regulatory transcription factors (TFs) to their sites on DNA and recruitment of the transcriptional machinery. Here we have analyzed changes in nucleosome occupancy at the enhancers as well as at the promoters of three pro-inflammatory genes when they are induced by bacterial lipopolysaccharides (LPS) in primary mouse macrophages. We find that nucleosomes are removed from the distal enhancers of IL12B and IL1A, as well as from the distal and proximal enhancers of IFNB1, and that clearance of enhancers correlates with binding of various cis-regulatory TFs. We further show that for IFNB1 the degree of nucleosome removal correlates well with the level of induction of the gene under different conditions. Surprisingly, we find that nucleosome occupancy at the promoters of IL12B and IL1A does not change significantly when the genes are induced, and that a considerably fraction of the cells is occupied by nucleosomes at any given time. We hypothesize that competing nucleosomes at the promoters of IL12B and IL1A may play a role in limiting the size of transcriptional bursts in individual cells, which may be important for controlling cytokine production in a population of immune cells.

Transcriptional reprogramming and chromatin remodeling accompanies Oct4 and Nanog silencing in mouse trophoblast lineage.

In mouse blastocysts, CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. However, the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression are poorly understood. To address this, a Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system. Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 h. Consistent with the down-regulation of Oct4 and Nanog transcripts, a time-dependent increase in CDX2 binding and a decrease in RNA polymerase II (RNAPII) and OCT4 binding was observed within 48 h (P<0.05). To test whether transcriptionally active epigenetic marks were erased during differentiation, histone H3K9/14 acetylation and two of its epigenetic modifiers were evaluated. Accordingly, a significant decrease in histone H3K9/14 acetylation and loss of p300 and HDAC1 binding at the Oct4 and Nanog regulatory elements was observed by 48 h. Accompanying these changes, there was a significant increase in total histone H3 and a loss of chromatin accessibility at both the Oct4 and Nanog regulatory elements (P<0.05), indicative of chromatin remodeling. Lastly, DNA methylation analysis revealed that methylation did not occur at Oct4 and Nanog until 96 to 120 h after induction of CDX2. In conclusion, our results show that silencing of Oct4 and Nanog is facilitated by sequential changes in transcription factor binding, histone acetylation, chromatin remodeling, and DNA methylation at core regulatory elements.

An effect of DNA sequence on nucleosome occupancy and removal.

A barrier phases nucleosomes at the yeast (Saccharomyces cerevisiae) GAL1-GAL10 genes. Here we separate nucleosome positioning from occupancy and show that the degree of occupancy of these phased sites is predictably determined by the underlying DNA sequences. As this occupancy is increased (by sequence alteration), nucleosome removal upon induction is decreased, as is mRNA production. These results explain why promoter sequences have evolved to form nucleosomes relatively inefficiently.