Bartholomew Laboratory

The Problem

The recent rise of the field of epigenetics has revealed that organismal development, cellular differentiation, and human disease depend not only on the DNA encoding individual genes, but also on how DNA is packaged into chromatin and how access to packaged DNA is regulated. One key class of epigenetic regulators is the ATP-dependent chromatin remodelers. This class comprises a large number of protein complexes that use the energy derived from ATP hydrolysis to remodel and/or reconfigure chromatin. These protein complexes vary in size from ~300 KDa to >1 MDa and belong to four major families: SWI/SNF, ISWI, CHD, and INO80. The challenge has been to define the similarities and differences between these complexes regarding how they reorganize chromatin and how they are recruited to different genomic sites. While we have learned some of the basic mechanisms related to these complexes, we have only begun to understand how individual proteins operate within the complexes and how mutations in these complexes lead to cancer and other diseases.

The Approach

The Bartholomew laboratory takes a multi-pronged approach to unraveling questions related to ATP-dependent chromatin remodelers and how they influence development and disease.  They start by analyzing the structure and function of these large chromatin remodeling machines using model systems like the yeast Saccharomyces cerevisiae, which provides an ideal platform for detailed biochemical and structural analyses. Structure/function relationships are typically tested by mutating or deleting individual protein domains within a subunit of the larger complex or within one or more of its targets followed by examining the activity of these complexes both in vitro and in vivo.  The information gained from the yeast analyses is then tested in mammalian systems using mouse embryonic stem cells (mESCs) in both their naïve state and when differentiated into various cell types. The cells are analyzed using a combination of cutting-edge genomics and state-of-the art microscopy to assess the impact of mutations on cellular activity (e.g. transcription) and chromatin architecture (e.g. chromatin structure and composition, and overall nuclear organization).  In addition, the Bartholomew lab frequently collaborates with experts who use specialized biophysical approaches such as single-molecule FRET and advanced mass spectrometry to probe structural dynamics and protein interaction networks

Nucleosome plasticity and the ISWI chromatin remodeler

For years, the nucleosome has been thought to be a rigid body that remains unchanged except for DNA movement, such as that occurring during ATP-dependent chromatin remodeling. The Bartholomew laboratory has recently provided compelling evidence that the histone octamer core of the nucleosome is not rigid, but rather it is dynamic. Significant distortions of the nucleosome core occur early during remodeling. When these distortions are restricted, no additional remodeling can occur. These studies focused on yeast ISW2 and employed a variety of approaches, including targeted mutagenesis, to trap remodeling intermediates and to identify when and how the histone octamer is distorted.  (For more information, see Hada et al., 2019, Cell Rep. )

Actin-related proteins (ARPs) in the INO80 complex serve as important sensors and regulators of nucleosome remodeling

INO80 is an ATP-dependent chromatin remodeler that not only moves nucleosomes on DNA but also exchanges histones out of nucleosomes, a process that is important for transcriptional regulation, DNA repair, and DNA replication. Nuclear actin and several actin-related proteins (ARP4, ARP5 and ARP8) are also present in the INO80 complex.

The Bartholomew laboratory discovered that ARP4, ARP8, and actin form a protein module that binds to extranucleosomal/linker DNA and positively regulates the nucleosome mobilizing activity of INO80 in a DNA length-dependent manner. Portions of ARP8 and ARP4 interact with DNA and actin whereas a portion of the catalytic subunit Ino80 serves as protein scaffold for these ARPs. They also discovered that ARP8 and ARP4 allosterically regulate the binding of ARP5 to the histone octamer, which is important for coupling ATPase activity to nucleosome movement. (For more information, see Brahma S, et al, 2018, Nat Commun.)        

The mammalian SWI/SNF complex promotes the exit from pluripotency by releasing paused RNA polymerase II via long-range chromatin interactions

In their newest research, the Bartholomew laboratory discovered that SWI/SNF releases RNA polymerase II (RNAPII) paused at developmental genes during the transition of mouse embryonic stem cells (mESCs) from their ground/naïve state to their primed/epiblast-like state. The release of RNAPII results from changes in long-range chromatin interactions between enhancers and gene promoters mediated by SWI/SNF. They also found that if these SWI/SNF mediated changes are blocked by mutating the SWIF/SNF catalytic subunit (SMARCA4/BRG1) to recreate mutations commonly found in cancer, then the differentiation of stem cells into cell types such as neural progenitor cells is altered.