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 inaccessible to most, but not all, 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. However, most of the studies establishing this view of chromatin and gene regulation were performed using isolated nuclei rather than live cells (see below). 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. 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 mutations in genes encoding 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 isolated 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, we have now established that isolated nuclei do not represent an accurate model for living cells, because chromatin dynamics are "frozen" when cells are disrupted. Disruption results in the loss of metabolites, including the ATP required for remodeling. We compared the accessibility of the genome in isolated nuclei and in living yeast cells using DNA methylases [1]. Remarkably, we found that the yeast genome is globally accessible in living cells, unlike in nuclei. That is, nucleosomes do not block access to genomic DNA in vivo. Briefly, we show 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 in living cells using an inducible promoter results in methylation of the entire genome, 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. The silenced mating type loci are also much less accessible than the rest of the genome. We show 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. However, budding yeast does not have the heterochromatin typical of the cells of higher organisms. Heterochromatin is generally associated with a high level of chromatin condensation and gene repression; it is expected to be much less accessible than transcriptionally active euchromatin. To address this important question, we extended our genome accessibility studies to human cell lines, specifically MCF7 breast cancer cells and MCF10A cells [2]. We observed that heterochromatin is methylated only slightly more slowly than euchromatin. That is, heterochromatin is also generally accessible in living cells. Again, we propose that ATP-dependent remodelers are responsible for maintaining DNA accessibility in live cells, and we invoke the same models for nucleosome dynamics that we proposed for yeast cells. 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. We continued our investigation of the roles of ATP-dependent chromatin remodelers in maintaining nucleosome dynamics in living yeast cells by focusing on the ISW1 and CHD1 nucleosome spacing enzymes [3,4]. We found that ISW1 and CHD1 together suppress nucleosome dynamics in living cells. That is, the genome is methylated faster in the absence of both remodeling enzymes. We propose that ISW1 and CHD1 slide nucleosomes to create an ordered spaced nucleosome array (i.e., the 'beads on a string') and suppress histone exchange (the cycle of histone removal from DNA and their replacement), resulting in reduced nucleosome dynamics and therefore in slower DNA methylation. In the absence of ISW1 and CHD1, their nucleosome sliding activities are lost but histone exchange increases, perhaps due to a nucleosome flux-creating remodeler, resulting in an overall increase in dynamics and methylation rate. Currently, we are testing various aspects of our nucleosome dynamics model in yeast and in human cell lines.