David Johannes Clark, Ph.D.

Aberrant gene regulation is the basis of many disease states. Our main objective is to understand how genes are activated for transcription in the context of chromatin structure. Chromatin is not just a packaging system for DNA in eukaryotic cells; it also participates in gene regulation. The structural subunit of chromatin is the nucleosome, which contains nearly two turns of DNA coiled around a central core histone octamer. Nucleosomes are generally quite regularly spaced along the DNA, like beads on a string. It is thought that gene regulation involves either attenuation of the inherently repressive properties of nucleosomes to facilitate gene expression, or enhancement of those properties to ensure complete repression. Gene regulation is choreographed by DNA sequence-specific transcription factors (activators and repressors) and chromatin remodeling complexes. The latter can be divided into two groups: histone or DNA modifying enzymes which implement the "epigenetic code", and ATP-dependent remodeling machines which move or displace nucleosomes. These enzymes are important because genes encoding remodeler subunits are often mutated in various cancers. Our recent work is focused on testing the hypothesis that nucleosomes control DNA accessibility and play a vital role in gene regulation by blocking promoters. We developed a method (qDA-seq) to obtain quantitative measurements of genome accessibility in nuclei and in living cells, using budding yeast and human cell lines. Our data show that chromatin is globally accessible in living yeast cells and in living human cells, but not in isolated nuclei. We propose that the ATP-dependent chromatin remodelers maintain most of the genome in an accessible state. We are currently exploring the ramifications of this surprising observation.

In parallel, we are studying DNA methylation patterns in human SDHx tumors. SDHx tumor cells contain excess succinate, resulting in inhibition of both DNA demethylases and histone demethylases. We are comparing DNA methylation patterns in SDHx tumor tissue with those in normal adrenal medulla tissue and in non-SDHx tumor tissue, using nanopore long-read DNA sequencing. The goal is to determine the genome-wide effects of excess succinate on DNA methylation.

Dr. David Clark has worked in the chromatin field since graduate school. He completed his PhD with Professor Jean Thomas in Cambridge. His studies addressed the mechanism of chromatin folding by linker histones. As a postdoctoral fellow in Dr. Gary Felsenfeld's lab at the NIH, Dr. Clark studied the problem of how RNA polymerase transcribes through a nucleosome. The nucleosome is a highly compact structure and a potent barrier to transcription. The lab was very interested in the proposal that a transcribing polymerase is preceded by positive supercoils with negative supercoils in its wake. Since the nucleosome contains negatively supercoiled DNA, it seemed possible that positive supercoils would destabilize nucleosomes ahead of the transcribing polymerase. It was found that nucleosomes are relatively unstable on positively supercoiled DNA and therefore possess a latent tendency for transfer to negatively supercoiled DNA. It was proposed that RNA polymerase might exploit this tendency during transcription: the nucleosome might be transferred from the positively supercoiled DNA in front of the polymerase to the negatively supercoiled DNA behind it. This idea was tested using a model system in which the fate of a single nucleosome placed at a defined position on a plasmid was determined after transcription. The nucleosome moved behind the transcribing polymerase. These observations were followed up in collaboration with Dr. Vasily Studitsky, resulting in a "spooling" model for transcription through the nucleosome. Independently, Dr. Clark published a paper with a postdoctoral colleague in Dr. Felsenfeld's lab, Dr. Takeshi Kimura, describing a theoretical analysis of chromatin folding using polyelectrolyte theory.

After establishing his own lab at the NIH, Dr. Clark developed a model system to study the events that occur when a gene is activated for transcription in vivo. Native plasmid chromatin containing a model gene expressed at basal or activated levels was purified from yeast cells. These studies revealed that activation correlates with large scale movements of nucleosomes and remodeling of nucleosomes over the entire gene. More recently, the Clark Lab has adopted global approaches made possible by massively parallel sequencing. The general consensus in the field is that chromatin regulates genes by blocking access to the DNA, unless activating ATP-dependent chromatin remodelers are recruited by transcription factors to gene regulatory elements, alleviating the block. This model is supported by many studies involving cell disruption prior to analysis (e.g., MNase-seq, ATAC-seq). However, these techniques do not provide an absolute measure of genomic DNA accessibility. The lab developed a quantitative assay for measuring DNA accessibility in yeast and mouse liver cell nuclei ("qDA-seq"). It was discovered that virtually all sites have limited accessibility; no sites are blocked in all nuclei, or accessible in all nuclei. Subsequently, qDA-seq was modified for use in vivo using inducible expression of the Dam DNA methylase (which methylates GATC sites) instead of a restriction enzyme. Remarkably, it was discovered that the entire genome is fully accessible in living yeast cells, except for the centromeres and the silenced mating type loci. It was concluded that yeast chromatin is globally dynamic in vivo, but static in nuclei. Consistent with these observations, the binding of the yeast Gcn4 transcription factor in vivo is determined primarily by site affinity, with chromatin contributing only a modest inhibitory effect. The lab went on to show that both euchromatin and heterochromatin are highly dynamic in human cells, using the MCF7 human breast cancer cell line and an adenovirus vector to express the Dam methylase. Current work focuses on identifying the remodelers responsible for nucleosome flux in vivo.