Hmo1 Protein Affects the Nucleosome Structure and Supports the Nucleosome Reorganization Activity of Yeast FACT.

Yeast Hmo1 is a high mobility group B (HMGB) protein that participates in the transcription of ribosomal protein genes and rDNA, and also stimulates the activities of some ATP-dependent remodelers. Hmo1 binds both DNA and nucleosomes and has been proposed to be a functional yeast analog of mammalian linker histones. We used EMSA and single particle Förster resonance energy transfer (spFRET) microscopy to characterize the effects of Hmo1 on nucleosomes alone and with the histone chaperone FACT.

Electron microscopy analysis of ATP-independent nucleosome unfolding by FACT.

FACT is a histone chaperone that participates in nucleosome removal and reassembly during transcription and replication. We used electron microscopy to study FACT, FACT:Nhp6 and FACT:Nhp6:nucleosome complexes, and found that all complexes adopt broad ranges of configurations, indicating high flexibility. We found unexpectedly that the DNA binding protein Nhp6 also binds to the C-terminal tails of FACT subunits, inducing more open geometries of FACT even in the absence of nucleosomes.

The interaction between the Spt6-tSH2 domain and Rpb1 affects multiple functions of RNA Polymerase II.

The conserved transcription elongation factor Spt6 makes several contacts with the RNA Polymerase II (RNAPII) complex, including a high-affinity interaction between the Spt6 tandem SH2 domain (Spt6-tSH2) and phosphorylated residues of the Rpb1 subunit in the linker between the catalytic core and the C-terminal domain (CTD) heptad repeats. This interaction contributes to generic localization of Spt6, but we show here that it also has gene-specific roles. Disrupting the interface affected transcription start site selection at a subset of genes whose expression is regulated by this choice, and this was accompanied by changes in a distinct pattern of Spt6 accumulation at these sites.

FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics.

Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes.

Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in .

FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone-DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity , and chromatin integrity and transcript output Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels.

Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization.

The essential histone chaperone FACT (cilitates hromatin ranscription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein.

Large-scale ATP-independent nucleosome unfolding by a histone chaperone.

DNA accessibility to regulatory proteins is substantially influenced by nucleosome structure and dynamics. The facilitates chromatin transcription (FACT) complex increases the accessibility of nucleosomal DNA, but the mechanism and extent of its nucleosome reorganization activity are unknown. Here we determined the effects of FACT from the yeast Saccharomyces cerevisiae on single nucleosomes by using single-particle Förster resonance energy transfer (spFRET) microscopy.

FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs.

FACT, a heterodimer of Spt16 and Pob3, is an essential histone chaperone. We show that the H2A-H2B binding activity that is central to FACT function resides in short acidic regions near the C termini of each subunit. Mutations throughout these regions affect binding and cause correlated phenotypes that range from mild to lethal, with the largest individual contributions unexpectedly coming from an aromatic residue and a nearby carboxylate residue within each domain.

The Abundant Histone Chaperones Spt6 and FACT Collaborate to Assemble, Inspect, and Maintain Chromatin Structure in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Spt6 protein is a conserved chromatin factor with several distinct functional domains, including a natively unstructured 30-residue N-terminal region that binds competitively with Spn1 or nucleosomes. To uncover physiological roles of these interactions, we isolated histone mutations that suppress defects caused by weakening Spt6:Spn1 binding with the spt6-F249K mutation. The strongest suppressor was H2A-N39K, which perturbs the point of contact between the two H2A-H2B dimers in an assembled nucleosome.

The FACT histone chaperone guides histone H4 into its nucleosomal conformation in Saccharomyces cerevisiae.

The pob3-Q308K mutation alters the small subunit of the Saccharomyces cerevisiae histone/nucleosome chaperone Facilitates Chromatin Transactions (FACT), causing defects in both transcription and DNA replication. We describe histone mutations that suppress some of these defects, providing new insight into the mechanism of FACT activity in vivo. FACT is primarily known for its ability to promote reorganization of nucleosomes into a more open form, but neither the pob3-Q308K mutation nor the compensating histone mutations affect this activity.

Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT.

The histone chaperone FACT is an essential and abundant heterodimer found in all eukaryotes. Here we report a crystal structure of the middle domain of the large subunit of FACT (Spt16-M) to reveal a double pleckstrin homology architecture. This structure was found previously in the Pob3-M domain of the small subunit of FACT and in the related histone chaperone Rtt106, although Spt16-M is distinguished from these structures by the presence of an extended α-helix and a C-terminal addition.

Insight into the mechanism of nucleosome reorganization from histone mutants that suppress defects in the FACT histone chaperone.

FACT (FAcilitates Chromatin Transcription/Transactions) plays a central role in transcription and replication in eukaryotes by both establishing and overcoming the repressive properties of chromatin. FACT promotes these opposing goals by interconverting nucleosomes between the canonical form and a more open reorganized form. In the forward direction, reorganization destabilizes nucleosomes, while the reverse reaction promotes nucleosome assembly.

Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication.

FACT plays important roles in both gene transcription and DNA replication. However, how this protein complex is targeted to these two distinct cellular processes remains largely unknown. Here we show that ubiquitylation of the Spt16 subunit of FACT by Rtt101, the cullin subunit of an E3 ubiquitin ligase in Saccharomyces cerevisiae, links FACT to DNA replication.

Breastfeeding in survivors of Hodgkin lymphoma treated with chest radiotherapy.

Background: Female survivors of therapeutic chest radiation often question whether they will have difficulty with breast feeding, given their prior exposure to breast radiation. In the current study, the rates of successful breastfeeding in long-term survivors who received chest radiotherapy (CXRT) as a treatment for Hodgkin lymphoma (HL) were examined.

Methods: A survey study of survivors of HL who were treated at the Joint Center for Radiation Therapy in Boston from 1969 through 1996 was performed.

Synthesis and characterization of an asymmetric, linear, trinuclear manganese(II) complex.

After prolonged heating in acetonitrile, a highly asymmetric, trinuclear manganous complex self-assembles from MnCl(2) and bis(2-pyridylmethyl)-1,2-ethanediamine (bispicen). The central Mn(II) ion is bridged to the terminal metal ions in the molecule by single chloride anions. The organic ligands each bind to a single Mn(II) ion.

yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement.

FACT has been proposed to function by displacing H2A-H2B dimers from nucleosomes to form hexasomes. Results described here with yeast FACT (yFACT) suggest instead that nucleosomes are reorganized to a form with the original composition but a looser, more dynamic structure. First, yFACT enhances hydroxyl radical accessibility and endonuclease digestion in vitro at sites throughout the nucleosome, not just in regions contacted by H2A-H2B.

Infantile lead poisoning from an Asian tongue powder: a case report & subsequent public health inquiry.

Introduction: Lead poisoning from novel environmental sources continues to present a challenge to clinicians who treat infants and children.

Case Report: A 12 month old infant of Thai parents was found during well child care to have a venous blood lead concentration of 61 mcg/dL. He was hospitalized for parenteral chelation with CaNa2EDTA and subsequently managed as an outpatient with oral succimer, with a reduction in blood lead concentration to 23 mcg/dL.

Structural and functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits.

yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site.

Improved retinal transduction in vivo and photoreceptor-specific transgene expression using adenovirus vectors with modified penton base.

Adenovirus (Ad) vectors can be injected into human ocular tissues without producing adverse events and are therefore a promising means of gene transfer to the retina. However, when administered subretinally, Ad vectors primarily transduce the retinal pigment epithelium (RPE), whereas the majority of mutant gene products that cause photoreceptor (PR) degeneration are expressed exclusively in the PR cells. While it has been shown previously that pseudotyping of Ad can partially overcome the limited PR transduction by Ad5, we found that pseudotyping of Ad is not necessary for transduction of PR cells.

blm3-1 is an allele of UBP3, a ubiquitin protease that appears to act during transcription of damaged DNA.

Yeast Blm10 and mammalian PA200 proteins share significant sequence similarity and both cap the ends of 20 S proteasomes and enhance degradation of some peptide substrates. Blm10 was identified as a suppressor of the yeast blm3-1 mutation, and initially was thought to be the Blm3 protein. Both the blm3-1 and blm10-Delta mutations were reported to cause sensitivity to bleomycin and other forms of DNA damage, suggesting a role for Blm10/PA200-proteasome complexes in DNA repair.