Chromatin Remodeling and Gene Activation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and transcription. These events coincide with the removal of promoter nucleosomes to create a nucleosome-depleted region (NDR). This observation has led to the generally accepted model that promoter nucleosomes physically block transcript initiation, acting as repressors by preventing access to specific transcription factor binding sites. The nucleosome is a very stable structure containing tightly wound DNA that is largely inaccessible to sequence-specific DNA binding proteins. Activation occurs if sequence-specific 'pioneer' transcription factors are present (these proteins bind nucleosomal sites with high affinity), and/or if 'classical' transcription factors, which are normally blocked by nucleosomes, recruit ATP-dependent chromatin remodelers to move or evict promoter nucleosomes, thus facilitating initiation complex formation. The ATP-dependent chromatin remodelers variously move nucleosomes along DNA, or remove the histones altogether, or form arrays of regularly spaced nucleosomes. Examples include the SWI/SNF and RSC complexes, which remodel nucleosomes on genes and at promoters, and the CHD and ISWI complexes, some of which are involved in determining nucleosome spacing. The INO80C complex is unusual because it appears to have both properties. Human diseases have been linked to chromatin remodeling enzymes. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors, and the CHD remodelers have been linked to cancer and autism. Therapies and drugs aimed at epigenetic targets are being tested. Thus, a full understanding of chromatin structure and the mechanisms by which it is manipulated is vital. The general view is that nucleosomes and chromatin structure play a central role in gene regulation by restricting access to genomic DNA. Experiments measuring DNA accessibility in nuclei have established that nucleosomes block access to DNA (e.g., MNase-seq, ATAC-seq, qDA-seq). Studies from many groups have confirmed this observation. It is stated as a fact in the introductions to most chromatin papers (including our own) and in reviews of the chromatin field. Indeed, the observation that nucleosomes block access to DNA is central to current models of gene regulation, as mentioned above. However, nuclei might not represent an accurate model for living cells, because chromatin dynamics may be "frozen" when cells are disrupted, which results in the loss of metabolites, such as the ATP required for remodeling. To address this question, we are comparing the accessibility of DNA genome-wide in nuclei and in living yeast cells using DNA methylases. Remarkably, we find that the genome is globally accessible in living cells, unlike in nuclei. That is, nucleosomes do not block access to genomic DNA in vivo. We find that methylation by the Dam DNA methylase (which methylates A in the sequence GATC) is blocked by nucleosomes in isolated nuclei. However, expression of Dam using an inducible promoter results in methylation of the entire genome in living cells, with minimal interference from nucleosomes. Using a different DNA methylase, M.SssI (which methylates C in CG), together with Nanopore long-read sequencing, we show that centromeric nucleosomes, unlike canonical nucleosomes, are exceptionally stable, protecting their DNA from methylation in vivo. We also observe that at least three ATP-dependent chromatin remodelers (RSC, ISW1 and CHD1) contribute to nucleosome dynamics in vivo, using a degron approach to detect nucleosome movements in living cells as remodelers are depleted. Our data demonstrate that nucleosomes are in a continuous state of flux in living cells, but static in nuclei, presumably due to loss of critical factors during isolation. This flux may involve nucleosome sliding, nucleosome removal and replacement and/or nucleosome conformational changes, catalyzed by ATP-dependent chromatin remodelers. We propose that the various remodelers compete with one another in vivo, continually moving nucleosomes to different positions, resulting in a nucleosome flux that renders the yeast genome essentially transparent to transcription factors and other DNA-binding proteins. Our observations have profound implications for the chromatin field, requiring a re-examination of the roles of the chromatin remodelers in gene regulation, and of the extent to which packaging the genome into nucleosomes is actually repressive. A manuscript describing these data is currently under review. We are extending our studies to human cell lines. Long-read sequencing methods, namely the PacBio and Nanopore platforms, are becoming increasingly popular as the power of these new technologies is fully appreciated. In our own field of chromatin biology, epigenetic modifications such as m5C and m6A can now be detected directly in original (i.e. unamplified) multi-kilobase DNA molecules. A recent extension of this approach is the use of methylation footprinting, in which an exogenous DNA methylase is added to nuclei or chromatin, where it methylates accessible regions, such as nucleosome-depleted promoters and the linkers between nucleosomes, but not nucleosomal DNA, which it cannot access. The result is a methylation map of each molecule, to be interpreted in terms of footprints and accessible regions. We have performed methylation footprinting in budding yeast nuclei using M.EcoGII, an adenine-specific DNA methylase. The potential footprint resolution of this approach is very high because of the adenine density of DNA. We used PacBio long-read sequencing to detect m6A introduced by M.EcoGII, and we developed a pipeline to analyze the data. We discovered a number of critical, previously unreported, issues concerning PacBio methylation footprinting data, which must be corrected before an accurate chromatin map can be obtained. Specifically, we observed low limit methylation levels in the genomic DNA positive controls, a wide range in the fraction of m6A from one DNA molecule to the next, and a strong local bias against methylation of AT-rich sequences and poly(A) runs. We also found that there is a high probability of observing a single m6A base within a nucleosome, breaking up the expected 147 bp footprint. Our novel probability model resolves all of these critical interpretative problems. The pipeline output includes an IGV-ready bam file, which displays both called m6A bases and our interpretation of the methylation pattern as accessible regions and nucleosomes in individual DNA molecules. We used our data to investigate heterogeneity in chromatin structure around the transcription start sites of yeast genes. We find that nucleosome positioning on a specific gene varies widely from cell to cell, with only a small fraction of genes showing a similar ordered nucleosomal array in every cell, even though the cell population average is as expected from other techniques, such as MNase-seq. We quantify the degree of heterogeneity for every yeast gene using a novel correlation score. A manuscript describing our data is currently in revision.