Steroid Receptors Reprogram FoxA1 Occupancy through Dynamic Chromatin Transitions.

The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.

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.

Polycomb-repressed genes have permissive enhancers that initiate reprogramming.

Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.

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.

Analysis of individual remodeled nucleosomes reveals decreased histone-DNA contacts created by hSWI/SNF.

Chromatin remodeling enzymes use the energy of ATP hydrolysis to alter histone-DNA contacts and regulate DNA-based processes in eukaryotes. Whether different subfamilies of remodeling complexes generate distinct products remains uncertain. We have developed a protocol to analyze nucleosome remodeling on individual products formed in vitro. We used a DNA methyltransferase to examine DNA accessibility throughout nucleosomes that had been remodeled by the ISWI and SWI/SNF families of enzymes. We confirmed that ISWI-family enzymes mainly created patterns of accessibility consistent with canonical nucleosomes. In contrast, SWI/SNF-family enzymes generated widespread DNA accessibility. The protection patterns created by these enzymes were usually located at the extreme ends of the DNA and showed no evidence for stable loop formation on individual molecules. Instead, SWI/SNF family proteins created extensive accessibility by generating heterogeneous products that had fewer histone-DNA contacts than a canonical nucleosome, consistent with models in which a canonical histone octamer has been 'pushed' off of the end of the DNA.

Complex genomic interactions in the dynamic regulation of transcription by the glucocorticoid receptor.

The glucocorticoid receptor regulates transcriptional output through complex interactions with the genome. These events require continuous remodeling of chromatin, interactions of the glucocorticoid receptor with chaperones and other accessory factors, and recycling of the receptor by the proteasome. Therefore, the cohort of factors expressed in a particular cell type can determine the physiological outcome upon treatment with glucocorticoid hormones. In addition, circadian and ultradian cycling of hormones can also affect GR response. Here we will discuss revision of the classical static model of GR binding to response elements to incorporate recent findings from single cell and genome-wide analyses of GR regulation. We will highlight how these studies have changed our views on the dynamics of GR recruitment and its modulation of gene expression.

High-throughput fluorescence-based screen to identify factors involved in nuclear receptor recruitment to response elements.

The glucocorticoid receptor is an inducible transcription factor which plays important roles in many -physiological processes. Upon activation, GR interacts with regulatory elements and modulates the expression of genes. Although GR is widely expressed in multiple tissues, its binding sites within chromatin and the genes it regulates are tissue specific. Many accessory proteins and cofactors are thought to play a role in dictating GR's function; however, mechanisms involved in targeting GR to specific sites in the genome are not well understood. Here we describe a high-throughput fluorescence-based method to identify factors involved in GR loading at response elements. This screen utilizes a genetically engineered cell line that contains 200 repeats of a glucocorticoid response promoter and expresses GFP-tagged GR. Upon treatment with corticosteroids, GFP-GR forms a steady-state distribution at the promoter array, and its concentration at this focal point can be quantitatively determined. This system provides a novel approach to identify activities important for GR loading at its response element using siRNA libraries to target factors that enhance or inhibit receptor localization.

Reprogramming the chromatin landscape: interplay of the estrogen and glucocorticoid receptors at the genomic level.

Cross-talk between estrogen receptors (ER) and glucocorticoid receptors (GR) has been shown to contribute to the development and progression of breast cancer. Importantly, the ER and GR status in breast cancer cells is a significant factor in determining the outcome of the disease. However, mechanistic details defining the cellular interactions between ER and GR are poorly understood. We investigated genome-wide binding profiles for ER and GR upon coactivation and characterized the status of the chromatin landscape. We describe a novel mechanism dictating the molecular interplay between ER and GR. Upon induction, GR modulates access of ER to specific sites in the genome by reorganization of the chromatin configuration for these elements. Binding to these newly accessible sites occurs either by direct recognition of ER response elements or indirectly through interactions with other factors. The unveiling of this mechanism is important for understanding cellular interactions between ER and GR and may represent a general mechanism for cross-talk between nuclear receptors in human disease.

Control of nuclear receptor function by local chromatin structure.

Steroid hormone receptors regulate gene transcription in a highly tissue-specific manner. The local chromatin structure underlying promoters and hormone response elements is a major component involved in controlling these highly restricted expression patterns. Chromatin remodeling complexes, as well as histone and DNA modifying enzymes, are directed to gene-specific regions and create permissive or repressive chromatin environments. These structures further enable proper communication between transcription factors, co-regulators and basic transcription machinery. The regulatory elements active at target genes can be either constitutively accessible to receptors or subject to rapid receptor-dependent modification. The chromatin states responsible for these processes are in turn determined during development and differentiation. Thus access of regulatory factors to elements in chromatin provides a major level of cell selective regulation.

Methylation-sensitive single-molecule analysis of chromatin structure.

Methylation-sensitive single-molecule analysis of chromatin structure is a high-resolution method for studying nucleosome positioning. As described in this unit, this method allows for the analysis of the chromatin structure of unmethylated CpG islands or in vitro-remodeled nucleosomes by treatment with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by bisulfite sequencing of individual progeny DNA molecules. Unlike nuclease-based approaches, this method allows each molecule to be viewed as an individual entity instead of an average population.