Bradley Cairns is interested in how chromatin structure helps regulate gene transcription. His lab purifies and characterizes large protein complexes that use ATP hydrolysis to remodel and modify chromosomal structure to regulate transcription. Connected to this, the lab also investigates how chromatin structure and transcription factors work together to control transcription in germline stem cells (which produce sperm and eggs) and early vertebrate embryos. He and his colleagues use genetic, biochemical, and genomic methods to understand the functions of these chromatin-regulatory complexes in living cells.
Chromosomal DNA is packaged into protein/DNA structures called chromatin. Certain chromatin structures silence genes, whereas others facilitate gene expression. Thus, chromatin is a remarkably dynamic material; structures formed to prevent gene expression can be remodeled to enable expression in response to cellular signals during growth and development. As many genes important for cell proliferation and differentiation are regulated at the chromatin level, it is important to understand how packaging by chromatin influences transcription, to characterize the protein machines that facilitate the transitions between chromatin states, and to understand how these machines are regulated. These are the goals of my laboratory.
The basic unit of chromatin structure is the nucleosome, which consists of a "bead" composed of eight histone proteins, around which 147 base pairs of DNA are wrapped. Nucleosomes display three dynamic properties in vivo: compositional changes, covalent modifications by enzymes, and repositioning (Figure 1). In regard to composition, nucleosomes can be reconstructed from canonical histones or from special histone variants. These variant nucleosomes assist with biological processes such as centromere construction or gene activation. Between nucleosomes is a small segment of free DNA (about 40 bases), leading to a view of the chromosome that resembles beads on a string. The wrapping of DNA around nucleosomes blocks the access of other protein factors to this DNA, and this can silence gene expression and other chromosomal processes. Another mode of gene silencing involves the methylation of the DNA itself—a covalent modification that recruits gene-silencing factors. Thus, DNA wrapping and modification work together to promote gene silencing and must be reversed for gene activation.
Cairns Research Abstract Slideshow
Figure 1: The three dynamic properties of nucleosomes. Changes in composition result from the action of histone-exchange complexes, such as the SWR1 complex, which replace canonical histones with a histone variant protein. These variants can attract special regulators (Reg), which affect the biology of the region. For example, histone H2A is replaced by the histone H2A variant Htz1 (green). Modification occurs on lysine residues on the amino terminal "tails" and can include acetylation (Ac), which is recognized by regulatory proteins. Repositioning of nucleosomes, which results in the exposure of DNA to transcriptional regulatory proteins, is performed by remodeler complexes.
Adapted from Saha, A. et al. 2006 Nature Reviews Molecular Cell Biology 7:437–447.
Figure 2: The remodeler ATPase subunit is divided into torsion and tracking subdomains that go through a series of movements (inchworm-like) to break histone-DNA contacts (orange, intact; yellow, broken) and enable directional sliding. Four steps are proposed: Step 1, the remodeler binds the nucleosome core, with the ATPase domain engaging the DNA about two turns from the dyad. Step 2, an ATP-dependent conformational change within the translocation domain causes the torsion subdomain to pull DNA from the linker into the nucleosome. This pulling places additional DNA in the cleft between the torsion and tracking subdomain, creating a DNA wave. Step 3, the tracking subdomain uses one of the two strands of the DNA as the tracking strand and allows the DNA wave to pass through it in a 3' to 5' direction. Step 4, the DNA wave propagates around the nucleosome by one-dimensional diffusion, breaking histone-DNA contacts at the leading edge and re-forming at the lagging edge. Following these steps, the torsion domain resets for an additional round of translocation.
Adapted from Saha, A. et al. 2006 Nature Reviews Molecular Cell Biology 7:437–447.
To enable access to the underlying DNA, nucleosomes must be mobilized, restructured, or ejected. As nucleosomes themselves have limited mobility, their dynamic movement requires the action of nucleosome-remodeling complexes (remodelers). Remodeler complexes use the energy from ATP hydrolysis to restructure, reposition, and eject nucleosomes, thus providing access to the DNA. Notably, gene activation also requires the lack of DNA methylation marks at the beginning of the gene (the promoter), which may involve either resistance to or removal of DNA methylation.
The Cairns lab is addressing several central questions in chromatin biology. First, we want to understand how remodeler complexes harness ATP hydrolysis to slide/move and eject nucleosomes, and how they know whether to slide or eject nucleosomes. Second, we want to understand how genes are selected to resist or receive DNA methylation (which causes silencing) and how DNA the methylation process (and other chromatin properties) are reprogrammed during development and cancer. We are especially interested in how this process is regulated in germline development and during embryo development, where genes turn on and off to change cell fate. Third, we examine how site-specific transcription factors interact with chromatin enzymes to help regulate chromatin reprogramming (and thus gene transcription) during germline and embryo development. Fourth, we study the transcription and modification of noncoding RNAs transcribed by RNA polymerase III (Pol III)—to understand how gene expression is controlled both by chromatin and by cellular and environmental conditions, and how RNA modifications are imposed on these noncoding RNAs.
To understand the first question - the mechanism that remodelers use to move or eject nucleosomes - we purify remodeler complexes and examine their action on purified nucleosomes in vitro. The primary remodeler that we study is RSC (remodels the structure of chromatin), which consists of 15 proteins in one large complex, that slides and ejects nucleosomes to help transcription factors bind DNA. We also work on the 'alternative' type of remodeler, termed ISWI, which helps organize and properly space nucleosomes to block transcription factor binding. We have shown that for both remodelers, the remodeler "engine" is an ATPase subunit that functions as a DNA translocase that pumps DNA around the surface of the nucleosome in the form of DNA waves (Figure 2), resulting in the movement of the histone octamer relative to the DNA. We have collaborated with Eva Nogales (HHMI, University of California, Berkeley) and Andres Leschziner (UC San Diego) to determine the structure of the entire RSC complex at moderate resolution, which revealed a large flexible protein machine that contains a large pocket of nucleosome dimensions. We have collaborated with Carlos Bustamante (HHMI, UC Berkeley) and Yongli Zhang (Yale University) to determine the speed and force of DNA translocation on individual RSC-nucleosome complexes. Cedric Clapier, Margared Kasten, Naveen Verma and Tim Mulvihill in my lab are studying how the ATPase subunit is regulated by RSC proteins and histone epitopes. Of particular interest are two actin-related proteins (ARPs), Arp7 and Arp9, bind directly to the ATPase subunit of the RSC remodeler and regulate the activity of the nucleosome-remodeling reaction. Clapier has developed new assays to monitor DNA translocation in a single-molecule format and to monitor nucleosome ejection. These assays are providing important details on how the translocase domain pumps DNA and how ARPs impact that mechanism. Over the past several years we have shown how the regulation of ATPase activity and the DNA translocation process (by ARPs) causes the alternative outcomes of nucleosome organization/spacing, regulated sliding, or ejection. (Clapier and Cairns, Nature 2012; Clapier et al., Molecular Cell, 2016, in press). We have also examined how the two complexes, RSC and ISWI, antagonize each other in vivo at gene promoters to help regulate transcription (Parnell, Schlichter et al., eLife 2015). We are now exploring how histone modifications/variants and transcription factors influence the activities of these remodelers.
Another major area of interest in our laboratory is how chromatin modifications help guide development of germline stem cells, gametogenesis (sperm and egg) and early embryo development. Our work in this area began with the first genome-wide examination of histone retention and modification in human sperm (Hammoud et al., Nature 2009). Remarkably, we have found that genes of developmental importance are packaged/poised in a distinctive manner in the germline (sperm), a packaging that is likely to promote their proper regulation in the embryo. We then turned to zebrafish to address many mechanistic questions of interest, which are technically impossibly in humans to address. Patrick Murphy, Magda Potok, Mengyao Tan, Yixuan Guo and Candice Wike are using the zebrafish model system to study the dynamic chromatin structure of germ cells, zygotes, and early embryos, and their interplay withe transcription factors (Potok et al., Cell 2013). Cairns lab members Jingtao Guo and Chongil Yi are also working (in collaboration with Sue Hammoud (Univ. of Michigan) and Douglas Carrell and Jim Hotaling, (University of Utah) on studying the chromatin status of mouse and human germline stem cells to better understand how these cells prepare the genome for later embryo development, with implications for male human fertility. A major emerging interest in the lab is how the human embryo employs a collaboration between transcription and chromatin factors to achieve 'totipotency' - the ability to become any embryonic or extraembryonic cell type - an effort involving lab members Pete Hendrickson, Jessie Dorais, and Ed Grow.
Finally, in addition to epigenetics, we are working on understanding the 'epitranscriptome' - modifications placed on noncoding RNAs that affect their function. Most noncoding RNAs are transcribed by RNA Polymerase III, and we determined the entire RNA Pol III transcriptome in human cells and have revealed the close collaboration between RNA Pol II (which transcribes mRNAs) enhancer-binding factors and Pol III: enhancer-binding factors open up the chromatin to allow Pol III access to Pol III genes, which reside in these enhancer (Oler et al., Nature Str. Mol Biol. 2010). We then developed an effective method to determine the locations of one of the most prevalent RNA modifications (cytosine methylation; Khoddami and Cairns, Nature Biotechnology 2013). We are now working on additional methods to profile additional major modifications at base-pair resolution.
This work is supported in part by grants from the National Institutes of Health.
As of April 25, 2016