Geeta J. Narlikar

Cooperation between complexes that regulate chromatin structure and transcription.

Chromatin structure creates barriers for each step in eukaryotic transcription. Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus.

Reconstitution of a Core Chromatin Remodeling Complex from SWI/SNF Subunits

Protein complexes of the SWI/SNF family remodel nucleosome structure in an ATP-dependent manner. Each complex contains between 8 and 15 subunits, several of which are highly conserved between yeast, Drosophila, and humans. We have reconstituted an ATP-dependent chromatin remodeling complex using a subset of conserved subunits. Unexpectedly, both BRG1 and hBRM, the ATPase subunits of human SWI/SNF complexes, are capable of remodeling mono-nucleosomes and nucleosomal arrays as purified proteins. The addition of INI1, BAF155, and BAF170 to BRG1 increases remodeling activity to a level comparable to that of the whole hSWI/SNF complex. These data define the functional core of the hSWI/SNF complex.

The Site-Specific Installation of Methyl-Lysine Analogs into Recombinant Histones

Histone lysine residues can be mono-, di-, or trimethylated. These posttranslational modifications regulate the affinity of effector proteins and may also impact chromatin structure independent of their role as adaptors. In order to study histone lysine methylation, particularly in the context of chromatin, we have developed a chemical approach to install analogs of methyl lysine into recombinant proteins. This approach allows for the rapid generation of large quantities of histones in which the site and degree of methylation can be specified. We demonstrate that these methyl-lysine analogs (MLAs) are functionally similar to their natural counterparts. These methylated histones were used to examine the influence of specific lysine methylation on the binding of effecter proteins and the rates of nucleosome remodeling. This simple method of introducing site-specific and degree-specific methylation into recombinant histones provides a powerful tool to investigate the biochemical mechanisms by which lysine methylation influences chromatin structure and function.

Mechanisms and functions of ATP-dependent chromatin-remodeling enzymes.

Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin. Here, we discuss such evidence and also relate mechanistic insights to our understanding of how chromatin-remodeling enzymes enable different in vivo processes.

Structural Basis of Silencing: Sir3 BAH Domain in Complex with a Nucleosome at 3.0 Å Resolution

A regulatory protein forms extensive interactions with the nucleosome core particle to create the basis for gene silencing. Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (Sir) proteins regulate mating type in Saccharomyces cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH (bromo-associated homology) domain and the nucleosome core particle at 3.0 angstrom resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4K16 and H3K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

Distinct Strategies to Make Nucleosomal DNA Accessible

One hallmark of ATP-dependent remodeling complexes is the ability to make nucleosomal DNA accessible to regulatory factors. We have compared two prominent human ATP-dependent remodelers, BRG1 from the SWI/SNF family and SNF2h from the ISWI family, for their abilities to make a spectrum of nucleosomal sites accessible. By measuring rates of remodeling at seven different sites on a mononucleosome and at six different sites on the central nucleosome of a trinucleosome, we have found that BRG1 opens centrally located sites more than an order of magnitude better than SNF2h. We provide evidence that this capability of BRG1 is caused by its ability to create DNA loops on the surface of a nucleosome, even when that nucleosome is constrained by adjacent nucleosomes. This specialized ability to make central sites accessible should allow SWI/SNF family complexes to facilitate binding of nuclear factors in chromatin environments where adjacent nucleosomes might otherwise constrain mobility.

Chromodomain-Mediated Oligomerization of HP1 Suggests a Nucleosome-Bridging Mechanism for Heterochromatin Assembly

HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9-methylated chromatin in vitro relies on an interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl mark recognition is highly sensitive to internucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. How the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. Here we show that nucleosome movement depends cooperatively on two ACF molecules, indicating that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one or both sides of the nucleosome. Three-dimensional reconstruction by single-particle electron microscopy of the ATPase–nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results indicate a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.

Dynamics of nucleosome remodelling by individual ACF complexes.

The ATP-dependent chromatin assembly and remodelling factor (ACF) functions to generate regularly spaced nucleosomes, which are required for heritable gene silencing. The mechanism by which ACF mobilizes nucleosomes remains poorly understood. Here we report a single-molecule FRET study that monitors the remodelling of individual nucleosomes by ACF in real time, revealing previously unknown remodelling intermediates and dynamics. In the presence of ACF and ATP, the nucleosomes exhibit gradual translocation along DNA interrupted by well-defined kinetic pauses that occurred after approximately seven or three to four base pairs of translocation. The binding of ACF, translocation of DNA and exiting of translocation pauses are all ATP-dependent, revealing three distinct functional roles of ATP during remodelling. At equilibrium, a continuously bound ACF complex can move the nucleosome back-and-forth many times before dissociation, indicating that ACF is a highly processive and bidirectional nucleosome translocase.

Generation and Interconversion of Multiple Distinct Nucleosomal States as a Mechanism for Catalyzing Chromatin Fluidity

We have dissected the steps in nucleosome remodeling by BRG1, the ATPase subunit of human SWI/SNF. BRG1-catalyzed DNA exposure is not enhanced by the proximity of the site to the ends of nucleosomal DNA, suggesting that the mechanism involves more than peeling or sliding of the DNA. Comparison of DNA exposure at specific sites with overall changes in the path of DNA implies that BRG1 generates multiple distinct remodeled structures and continuously interconverts them. These characteristics are shared by the entire SWI/SNF complex and have parallels, as well as interesting differences, with the activities of GroEL and Hsp70 protein chaperones. The chaperone-like activity of SWI/SNF is expected to create multiple opportunities for the binding of distinct regulatory factors, providing one mechanism by which SWI/SNF family complexes can contribute to both activation and repression of transcription.