Sam John

Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor–genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome.

Transcriptional activators direct histone acetyltransferase complexes to nucleosomes

Transcriptional co-activators were originally identified as proteins that act as intermediaries between upstream activators and the basal transcription machinery. The discovery that co-activators such as Tetrahymena and yeast Gcn5,, as well as human p300/CBP,, pCAF, Src-1, ACTR and TAFII250, can acetylate histones suggests that activators may be involved in targeting acetylation activity to promoters. Several histone deacetylases have been linked to transcriptional co-repressor proteins, suggesting that the action of both acetylases and deacetylases is important in the regulation of many genes. Here we demonstrate the binding of two native yeast histone acetyltransferase (HAT) complexes to the herpesvirus VP16 activation domain and the yeast transcriptional activator Gcn4, and show that it is their interaction with the VP16 activation domain that targets Gal4–VP16-bound nucleosomes for acetylation. We find that Gal4–VP16-driven transcription from chromatin templates is stimulated by both HAT complexes in an acetyl CoA-dependent reaction. Our results demonstrate the targeting of native HAT complexes by a transcription-activation domain to nucleosomes in order to activate transcription.

Expanded Lysine Acetylation Specificity of Gcn5 in Native Complexes

The coactivator/adaptor protein Gcn5 is a conserved histone acetyltransferase, which functions as the catalytic subunit in multiple yeast transcriptional regulatory complexes. The ability of Gcn5 to acetylate nucleosomal histones is significantly reduced relative to its activity on free histones, where it predominantly modifies histone H3 at lysine 14. However, the association of Gcn5 in multisubunit complexes potentiates its nucleosomal histone acetyltransferase activity. Here, we show that the association of Gcn5 with other proteins in two native yeast complexes, Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of acetylation, suggesting that the association of specific subunits determines site specificity.

Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding

Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.

Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription.

Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.

Interaction of the glucocorticoid receptor with the chromatin landscape

The generality and spectrum of chromatin-remodeling requirements for nuclear receptor function are unknown. We have characterized glucocorticoid receptor (GR) binding events and chromatin structural transitions across GR-induced or -repressed genes. This analysis reveals that GR binding invariably occurs at nuclease-accessible sites (DHS). A remarkable diversity of mechanisms, however, render these sites available for GR binding. Accessibility of the GR binding sites is either constitutive or hormone inducible. Within each category, some DHS sites require the Brg1-containing Swi/Snf complex, but others are Brg1 independent, implicating a different remodeling complex. The H2A.Z histone variant is highly enriched at both inducible and constitutive DHS sites and is subject to exchange during hormone activation. The DHS profile is highly cell specific, implicating cell-selective organization of the chromatin landscape as a critical determinant of tissue-selective receptor function. Furthermore, the widespread requirement for chromatin remodeling supports the recent hypothesis that the rapid exchange of receptor proteins occurs during nucleosome reorganization.

Actin-dependent intranuclear repositioning of an active gene locus in vivo

Although bulk chromatin is thought to have limited mobility within the interphase eukaryotic nucleus, directed long-distance chromosome movements are not unknown. Cajal bodies (CBs) are nuclear suborganelles that nonrandomly associate with small nuclear RNA (snRNA) and histone gene loci in human cells during interphase. However, the mechanism responsible for this association is uncertain. In this study, we present an experimental system to probe the dynamic interplay of CBs with a U2 snRNA target gene locus during transcriptional activation in living cells. Simultaneous four-dimensional tracking of CBs and U2 genes reveals that target loci are recruited toward relatively stably positioned CBs by long-range chromosomal motion. In the presence of a dominant-negative mutant of β-actin, the repositioning of activated U2 genes is markedly inhibited. This supports a model in which nuclear actin is required for these rapid, long-range chromosomal movements.

Extensive chromatin remodelling and establishment of transcription factor ‘hotspots’ during early adipogenesis

Adipogenesis is tightly controlled by a complex network of transcription factors acting at different stages of differentiation. Peroxisome proliferator‐activated receptor γ (PPARγ) and CCAAT/enhancer‐binding protein (C/EBP) family members are key regulators of this process. We have employed DNase I hypersensitive site analysis to investigate the genome‐wide changes in chromatin structure that accompany the binding of adipogenic transcription factors. These analyses revealed a dramatic and dynamic modulation of the chromatin landscape during the first hours of adipocyte differentiation that coincides with cooperative binding of multiple early transcription factors (including glucocorticoid receptor, retinoid X receptor, Stat5a, C/EBPβ and ‐δ) to transcription factor ‘hotspots’. Our results demonstrate that C/EBPβ marks a large number of these transcription factor ‘hotspots’ before induction of differentiation and chromatin remodelling and is required for their establishment. Furthermore, a subset of early remodelled C/EBP‐binding sites persists throughout differentiation and is later occupied by PPARγ, indicating that early C/EBP family members, in addition to their well‐established role in activation of PPARγ transcription, may act as pioneering factors for PPARγ binding.

Dynamic Exchange at Regulatory Elements during Chromatin Remodeling Underlies Assisted Loading Mechanism

The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.

Dynamic behavior of transcription factors on a natural promoter in living cells

Through the use of photobleaching techniques, we examined the dynamic interaction of three members of the transcrsiption apparatus with a target promoter in living cells. The glucocorticoid receptor (GR) interacting protein 1 (GRIP‐1) exhibits a half maximal time for fluorescent recovery (τR) of 5 s, reflecting the same rapid exchange as observed for GR. In contrast, the large subunit (RPB1) of RNA polymerase II (pol II) required 13 min for complete fluorescence recovery, consistent with its function as a processive enzyme. We also observe a complex induction profile for the kinetics of GR‐stimulated transcription. Our results indicate that GR and GRIP‐1 as components of the activating complex are in a dynamic equilibrium with the promoter, and must return to the template many times during the course of transcriptional activation.