Jan Wisniewski

Nonhistone Scm3 and Histones CenH3-H4 Assemble the Core of Centromere-Specific Nucleosomes

The budding yeast histone H3 variant, Cse4, replaces conventional histone H3 in centromeric chromatin and, together with centromere-specific DNA-binding factors, directs assembly of the kinetochore, a multiprotein complex mediating chromosome segregation. We have identified Scm3, a nonhistone protein that colocalizes with Cse4 and is required for its centromeric association. Bacterially expressed Scm3 binds directly to and reconstitutes a stoichiometric complex with Cse4 and histone H4 but not with conventional histone H3 and H4. A conserved acidic domain of Scm3 is responsible for directing the Cse4-specific interaction. Strikingly, binding of Scm3 can replace histones H2A-H2B from preassembled Cse4-containing histone octamers. This incompatibility between Scm3 and histones H2A-H2B is correlated with diminished in vivo occupancy of histone H2B, H2A, and H2AZ at centromeres. Our findings indicate that nonhistone Scm3 serves to assemble and maintain Cse4-H4 at centromeres and may replace histone H2A-H2B dimers in a centromere-specific nucleosome core.

Fast multicolor 3D imaging using aberration-corrected multifocus microscopy.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin

The Drosophila nucleosome remodeling factor (NURF) is a protein complex of four subunits that assists transcription factor-mediated perturbation of nucleosomes in an ATP-dependent manner. We have investigated the role of NURF in activating transcription from a preassembled chromatin template and have found that NURF is able to facilitate transcription mediated by a GAL4 derivative carrying both a DNA binding and an activator domain. Interestingly, once nucleosome remodeling by the DNA binding factor is accomplished, a high level of NURF activity is not continuously required for recruitment of the general transcriptional machinery and transcription for at least 100 nucleotides. Our results provide direct evidence that NURF is able to assist gene activation in a chromatin context, and identify a stage of NURF dependence early in the process leading to transcriptional initiation.

Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres

The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3. DOI: http://dx.doi.org/10.7554/eLife.02203.001

Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

Significance A major challenge in modern biological studies is in the determination of the 3D molecular architecture of cellular organelles. In recent years, much progress in nanoscale imaging has been made because of the advent of superresolution optical microscopy. However, many superresolution techniques are still limited to 2D acquisition. Here, we show a volumetric approach for superresolution imaging based on the simultaneous imaging of multiple sample planes using multifocal microscopy. The depth over which structures can be reconstructed reaches 4 µm, comparable with the thickness of many cellular organelles or even whole cells. Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division.

Purification of heat shock transcription factor of Drosophila

Publisher Summary This chapter discusses the purification of heat-shock transcription factor of Drosophila. All living organisms respond to elevated temperatures, and to a variety of chemical and physiological stresses by a rapid and transient increase in the synthesis of heat-shock proteins. These proteins function as molecular chaperones involved in protein folding, protein translocation, higher order assembly, and protein degradation. In eukaryotes, transcriptional regulation of the heat-shock genes is under the control of a conserved regulator referred to as the heat-shock transcription factor (HSF). HSF acts through the highly conserved heat-shock response element (HSE), composed of three inverted repeats of the 5-bp sequence nGAAn. HSF is present in a latent state under normal conditions and becomes activated as a consequence of heat stress. Activation of HSF generally occurs through two stages: (1) the induction of high-affinity binding to heat-shock promoters, accomplished by trimerization and cooperative interactions between HSF trimers and (2) the exposure of one or more activator domains. The chapter provides protocols for the purification of the endogenous and recombinant HSF proteins.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

16 Structure and Regulation of Heat Shock Transcription Factor

I. INTRODUCTION The transcriptional induction of heat shock genes by elevated temperatures and other forms of physiological stress in eukaryotes is mediated by the transcription factor heat shock factor (HSF). Considerable progress in our understanding of the structure of HSF and how its activity is regulated by heat shock has been achieved since the identification and purification of HSF proteins from a variety of metazoan species. For a review of the early studies, see Lis et al. (1990) and Wu et al. (1990). Here, we summarize recent advances in the structure and regulation of HSF, drawing primarily from studies in our laboratory on the Drosophila and human HSF proteins. Further elaborations on HSF, including the multiplicity of HSF genes and how heat shock promoters are poised to respond to HSF binding, are treated elsewhere in this volume (see Sarge et al.; Lis et al.). II. DNA BINDING BY A MONOMER-TRIMER TRANSITION Like many inducible transcriptional regulators, the HSF protein is synthesized constitutively and stored in a latent form under normal conditions. This property of HSF, originally observed for human and Drosophila HSF proteins (Kingston et al. 1987; Zimarino and Wu 1987), appears to be common to all eukaryotic species studied. With the exception of budding yeasts, the latent HSF is activated in response to heat shock by the acquisition of high-affinity DNA-binding activity, which is accomplished by a conversion of HSF protein from a monomer to a homotrimer (Perisic et al. 1989; Westwood et al. 1991; Baler et al. 1993;...