Structural basis of nucleosome transcription mediated by Chd1 and FACT.

Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT. In the first structure, Pol II transcription exposes the proximal histone H2A-H2B dimer that is bound by Spt5. Pol II has also released the inhibitory DNA-binding region of Chd1 that is poised to pump DNA toward Pol II. In the second structure, Pol II has generated a partially unraveled nucleosome that binds FACT, which excludes Chd1 and Spt5. These results suggest that Pol II progression through a nucleosome activates Chd1, enables FACT binding and eventually triggers transfer of FACT together with histones to upstream DNA.

Promotion of Hyperthermic-Induced rDNA Hypercondensation in Saccharomyces cerevisiae.

Ribosome biogenesis is tightly regulated through stress-sensing pathways that impact genome stability, aging and senescence. In Saccharomyces cerevisiae, ribosomal RNAs are transcribed from rDNA located on the right arm of chromosome XII. Numerous studies reveal that rDNA decondenses into a puff-like structure during interphase, and condenses into a tight loop-like structure during mitosis. Intriguingly, a novel and additional mechanism of increased mitotic rDNA compaction (termed hypercondensation) was recently discovered that occurs in response to temperature stress (hyperthermic-induced) and is rapidly reversible. Here, we report that neither changes in condensin binding or release of DNA during mitosis, nor mutation of factors that regulate cohesin binding and release, appear to play a critical role in hyperthermic-induced rDNA hypercondensation. A candidate genetic approach revealed that deletion of either HSP82 or HSC82 (Hsp90 encoding heat shock paralogs) result in significantly reduced hyperthermic-induced rDNA hypercondensation. Intriguingly, Hsp inhibitors do not impact rDNA hypercondensation. In combination, these findings suggest that Hsp90 either stabilizes client proteins, which are sensitive to very transient thermic challenges, or directly promotes rDNA hypercondensation during preanaphase. Our findings further reveal that the high mobility group protein Hmo1 is a negative regulator of mitotic rDNA condensation, distinct from its role in promoting premature condensation of rDNA during interphase upon nutrient starvation.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology.

The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA.

The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided evidence of cross-linked dimers, trimers and tetramers of xl-mtTFA. Finally, high resolution scanning transmission electron microscopy (STEM) established that xl-mtTFA binds to the specific promoter-proximal site predominantly as a tetramer. Computer analysis of several previously characterized binding sites for xl-mtTFA revealed a fine structure consisting of two half-sites in a symmetrical orientation. The predominant sequence of this dyad symmetry motif shows homology to binding sites of sequence-specific HMG-box-containing proteins such as Sry and Lef-1. We suggest that bidirectional activation of transcription results from the fact that binding of a tetramer of xl-mtTFA permits symmetrical interactions with other components of the transcription machinery at the adjacent core promoters.

SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein.

SARs are candidate DNA elements for defining the bases of chromatin loops and possibly for serving as cis elements of chromosome dynamics. SARs contain numerous A tracts, whose altered DNA structure is recognized by cooperatively interacting proteins such as topoisomerase II. We constructed multi-AT hook (MATH) proteins and demonstrate that they specifically bind the clustered A tracts of SARs in chromatin and chromosomes. They are also potent inhibitors of chromosome assembly in mitotic Xenopus extracts, demonstrating the importance of SARs in this process. Titration of SARs with MATH20 (20 hooks) blocks shape determination of chromatids but not chromatin condensation per se. SARs are also required for shape maintenance of chromosomes. If MATH20 is added after formation of chromatids, they collapse and are reshaped by an active, mitotic process into spherical chromatid balls.

The HMG-14/-17 chromosomal protein family: architectural elements that enhance transcription from chromatin templates.

Chromosomal proteins HMG-14 and HMG-17 enhance the transcriptional potential of chromatin when incorporated into nucleosomes during, but not after, chromatin assembly on replicating DNA. Two molecules of either HMG-14 or HMG-17 can bind to nucleosome cores, independently of the underlying DNA sequence, in a cooperative fashion to limit nucleosome mobility and stabilize the structure of the nucleosome core without stabilizing the higher order chromatin structure. By modifying the structure of nucleosomes, the proteins affect the local structure of the chromatin fiber leading to an increase in the rate of transcriptional elongation but not initiation. We suggest that HMG-14/-17 are architectural elements which assist in the assembly of an unfolded chromatin fiber thereby decreasing the repressive activity of histones and facilitating transcriptional processes.

Interaction between HMG proteins (1 + 2) and the negative regulatory region 1 (NCR1) in the 5'-flanking sequence of the human beta-globin gene.

The pattern of high mobility group proteins 1 and 2 (HMG1,2) interaction with the 5'-flanking sequence of the human beta-globin gene has been analyzed by scanning tunnelling microscopy (STM). A 200 bp negative regulatory region in the 5'-flanking sequence of the human beta-globin gene can be folded by HMG proteins 1 and 2 into a circular structure (diameter 70 +/- 6 A) with a linear tail which seems to be a left-handed double helix structure.

Structure of the HMG box motif in the B-domain of HMG1.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.

Immunochemical analysis of the exposure of high mobility group protein 14 and 17 surfaces in chromatin.

Antisera were elicited against synthetic peptides corresponding either to regions common to all members of the high mobility group 14 and 17 protein family protein or to distinct domains of the HMG-14 or HMG-17 subgroup. The antisera were used to probe the accessibility of various HMG domains in chromatin. Competitive enzyme-linked immunosorbent assays indicate that the central region of the proteins, which contains their DNA binding domain and is positively charged, is exposed to a smaller degree than the C-terminal region of the proteins, which has a net negative charge. The C-terminal regions of the HMG-14 and HMG-17 proteins are exposed and available to interact with other proteins.

Isolation and characterization of folded fragments released by Staphylococcal aureus proteinase from the non-histone chromosomal protein HMG-1.

HMG-1 was isolated from newborn calf thymus without exposure to overt denaturing conditions. The purified protein was digested under several solvent conditions with the proteinase (endoproteinase GluC) from Staphylococcus aureus strain V8. We found that the preferred site of attack by the enzyme on HMG-1 was influenced markedly by ionic strength and temperature. In 0.35 M NaCl/50 mM Tris-phosphate (pH 7.8) at 37 degrees C, cleavage near the junction between the A and B domains is predominant, as previously reported by Carballo et al. (EMBO J. 2 (1983) 1759-1764). However, in 50 mM Tris-phosphate (pH 7.8) lacking NaCl and at 0 degrees C, cleavage between the B and C domains strongly predominates. Three major products of the digestions were purified and characterized. The fragment consisting of domains B and C was found by circular dichroism to contain a substantial amount of helix. This re-emphasizes the importance of avoiding overt denaturing conditions when working with members of the HMG-1 family.