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. Hmo1 induced a significant increase in the distance between the DNA gyres across the nucleosomal core, and also caused the separation of linker segments. This was opposite to the effect of the linker histone H1, which enhanced the proximity of linkers. Similar to Nhp6, another HMGB factor, Hmo1, was able to support large-scale, ATP-independent, reversible unfolding of nucleosomes by FACT in the spFRET assay and partially support FACT function in vivo. However, unlike Hmo1, Nhp6 alone does not affect nucleosome structure. These results suggest physiological roles for Hmo1 that are distinct from Nhp6 and possibly from other HMGB factors and linker histones, such as H1.

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. Nhp6 therefore supports nucleosome unfolding by altering both the structure of FACT and the properties of nucleosomes. Complexes formed with FACT, Nhp6, and nucleosomes also produced a broad range of structures, revealing a large number of potential intermediates along a proposed unfolding pathway. The data suggest that Nhp6 has multiple roles before and during nucleosome unfolding by FACT, and that the process proceeds through a series of energetically similar intermediate structures, ultimately leading to an extensively unfolded form.

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. Splicing efficiency was also diminished, as was apparent progression through introns that encode snoRNAs. Chromatin-mediated repression was impaired, and a distinct role in maintaining +1 nucleosomes was identified, especially at ribosomal protein genes. The Spt6-tSH2:Rpb1 interface therefore has both genome-wide functions and local roles at subsets of genes where dynamic decisions regarding initiation, transcript processing, or termination are made. We propose that the interaction modulates the availability or activity of the core elongation and histone chaperone functions of Spt6, contributing to coordination between RNAPII and its accessory factors as varying local conditions call for dynamic responses.

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. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.

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

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 in vitro, and chromatin integrity and transcript output in vivo 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. The results are consistent with emerging models that emphasize FACT's importance in establishing global chromatin architecture prior to transcription, promoting transitions among different states as transcription profiles change, and restoring chromatin integrity after it is disturbed. Optimal FACT activity required the availability of both modified and unmodified states of H3-K56. Perturbing this balance was especially detrimental for maintaining repression of genes with high nucleosome occupancy over their promoters and for blocking antisense transcription at the +1 nucleosome. The results reveal a complex collaboration between H3-K56 modification status and multiple FACT functions, and support roles for nucleosome reorganization by FACT before, during, and after transcription.

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

The essential histone chaperone FACT (facilitates chromatin transcription) 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. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

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 binding results in dramatic ATP-independent, symmetrical and reversible DNA uncoiling that affects at least 70% of the DNA within a nucleosome, occurs without apparent loss of histones and proceeds via an 'all-or-none' mechanism. A mutated version of FACT is defective in uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus, FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this activity is an important function of FACT in vivo.

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. Spt16 and Pob3 bind overlapping sites on H2A-H2B, and Spt16-Pob3 heterodimers simultaneously bind two H2A-H2B dimers, the same stoichiometry as the components of a nucleosome. An Spt16:H2A-H2B crystal structure explains the biochemical and genetic data, provides a model for Pob3 binding, and implies a mechanism for FACT reorganization that we confirm biochemically. Moreover, unexpected similarity to binding of ANP32E and Swr1 with H2A.Z-H2B reveals that diverse H2A-H2B chaperones use common mechanisms of histone binding and regulating nucleosome functions.

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. Substantial suppression also was observed when the H2A-H2B interface with H3-H4 was altered, and many members of this class of mutations also suppressed a defect in another essential histone chaperone, FACT. Spt6 is best known as an H3-H4 chaperone, but we found that it binds with similar affinity to H2A-H2B or H3-H4. Like FACT, Spt6 is therefore capable of binding each of the individual components of a nucleosome, but unlike FACT, Spt6 did not produce endonuclease-sensitive reorganized nucleosomes and did not displace H2A-H2B dimers from nucleosomes. Spt6 and FACT therefore have distinct activities, but defects can be suppressed by overlapping histone mutations. We also found that Spt6 and FACT together are nearly as abundant as nucleosomes, with ∼24,000 Spt6 molecules, ∼42,000 FACT molecules, and ∼75,000 nucleosomes per cell. Histone mutations that destabilize interfaces within nucleosomes therefore reveal multiple spatial regions that have both common and distinct roles in the functions of these two essential and abundant histone chaperones. We discuss these observations in terms of different potential roles for chaperones in both promoting the assembly of nucleosomes and monitoring their quality.

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. Instead, purified mutant FACT complexes fail to release from nucleosomes efficiently, and the histone mutations correct this flaw. We confirm that pob3-T252E also suppresses pob3-Q308K and show that combining two suppressor mutations can be detrimental, further demonstrating the importance of balance between association and dissociation for efficient FACT:nucleosome interactions. To explain our results, we propose that histone H4 can adopt multiple conformations, most of which are incompatible with nucleosome assembly. FACT guides H4 to adopt appropriate conformations, and this activity can be enhanced or diminished by mutations in Pob3 or histones. FACT can therefore destabilize nucleosomes by favoring the reorganized state, but it can also promote assembly by tethering histones and DNA together and maintaining them in conformations that promote canonical nucleosome formation.

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. Consistent with our finding that the double pleckstrin homology structure is common to these three histone chaperones and reports that Pob3 and Rtt106 double pleckstrin homology domains bind histones H3-H4, we also find that Spt16-M binds H3-H4 with low micromolar affinity. Our structure provides a framework for interpreting a large body of genetic data regarding the physiological functions of FACT, including the identification of potential interaction surfaces for binding histones or other proteins.

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. Nucleosome destabilization involves disrupting contacts among histone H2A-H2B dimers, (H3-H4)(2) tetramers, and DNA. Here we show that mutations that weaken the dimer:tetramer interface in nucleosomes suppress defects caused by FACT deficiency in vivo in the yeast Saccharomyces cerevisiae. Mutating the gene that encodes the Spt16 subunit of FACT causes phenotypes associated with defects in transcription and replication, and we identify histone mutants that selectively suppress those associated with replication. Analysis of purified components suggests that the defective version of FACT is unable to maintain the reorganized nucleosome state efficiently, whereas nucleosomes with mutant histones are reorganized more easily than normal. The genetic suppression observed when the FACT defect is combined with the histone defect therefore reveals the importance of the dynamic reorganization of contacts within nucleosomes to the function of FACT in vivo, especially to FACT's apparent role in promoting progression of DNA replication complexes. We also show that an H2B mutation causes different phenotypes, depending on which of the two similar genes that encode this protein are altered, revealing unexpected functional differences between these duplicated genes and calling into question the practice of examining the effects of histone mutants by expressing them from a single plasmid-borne allele.

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. We find Rtt101 interacts with and ubiquitylates Spt16 in vitro and in vivo. Deletion of RTT101 leads to reduced association of both FACT and the replicative helicase MCM with replication origins. Loss of Rtt101 also reduces binding of FACT to MCM, but not the association of FACT with Leo1 and Spt5, two proteins involved in transcription. Origin function is compromised in cells lacking Rtt101 or with an Spt16 mutation. These findings identify Spt16 as an Rtt101 substrate, and suggest that Spt16 ubiquitylation is important for FACT to function during 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. Patients were queried about childbearing and breastfeeding. The authors analyzed self-reported breastfeeding success in women who reported live births after CXRT and compared them with a sibling control group. RESULTS: Overall, 83 female survivors of HL who were treated with CXRT reported at least 1 birth after treatment. There were a total of 141 post-HL pregnancies resulting in births. In these women, the median age at the time of the HL diagnosis was 23 years (range, 14-40 years) and the median radiation dose was 41 grays (Gy) (range, 27-46 Gy). For survivors, 57 of the 94 (61%) breastfeeding attempts were successful, whereas within the control group, 74 of the 94 (79%) attempts were successful (P = .044). Accounting for maternal age at first birth and birth era, multivariable analysis suggested that HL survivors treated with CXRT may be less likely to breastfeed successfully than their siblings (odds ratio, 0.42; 95% confidence interval, 0.2-1.0 [P = .06]). CONCLUSIONS: The majority of female HL survivors who were treated with CXRT report being able to breastfeed successfully. However, the results of the current study indicate a trend toward less successful breastfeeding in survivors as a previously unreported late effect of radiation.

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. Accessibility increases dramatically, but the DNA remains partially protected. Second, increased nuclease sensitivity can occur without displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter during transcriptional activation in vivo, but the preferential reduction in dimer occupancy expected for hexasome formation is not observed. We propose that yFACT promotes a reversible transition between two nucleosomal forms, and that this activity contributes to the establishment and maintenance of the chromatin barrier as well as to overcoming it.

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. Chronic lead poisoning was attributed to the use of a Thai tongue powder by the parents for the first seven months of the infant's life. This ethnic remedy was applied to the tongue to absorb toxins, reduce white patches present after milk feedings, and preserve the infant's health. INVESTIGATIONS: Lead contaminated the powder at 109,000 ppm as measured by x-ray fluorescence spectrometry. Two poison centers in Thailand were contacted and initiated a public health inquiry with the Thai Food & Drug Administration (Thai FDA) to remove contaminated products from the marketplace. Their investigation found six additional contaminated tongue powders (of 10 tested) in a Bangkok shop offering Chinese remedies, some with lead levels > 9000 ppm. These products, unregistered with the Thai FDA, were confiscated and the shop closed. Local media attention and case-finding activities of health officials identified one additional infant suffering from lead poisoning due to tongue powders. CONCLUSIONS: Asian tongue powders can be a source of lead poisoning. Medical toxicologists, poison centers, and public health agencies can work together internationally to accomplish effective post-marketing product surveillance.

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. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.

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. We determined that, in the context of Ad, the cytomegalovirus (CMV) promoter is not significantly active in PRs. We compared expression levels from CMV and chicken beta actin (CBA) promoters in neural retina and found that CBA has a 173-fold greater potency than CMV. We also investigated the nature of the Ad-RPE interaction in murine retina and determined that the RGD domain in Ad penton plays a key role in RPE tropism. Deletion of the RGD domain coupled with use of the CBA promoter permitted transgene expression in neural retina approximately 667 times more efficiently than with Ad5 vectors. The use of these vectors in combination with a 4.7 kilobase (kb) rhodopsin promoter enabled transgene expression exclusively in PR cells in vivo.

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. We have been unable to observe significant DNA damage sensitivity in blm10-Delta mutants in several genetic backgrounds, and we have therefore further investigated the relationship between BLM10 and blm3-1. We find that blm3-1 is a nonsense mutation in the ubiquitin protease gene UBP3. Deleting UBP3 causes phenotypes similar to those caused by blm3-1, but neither causes a general defect in DNA repair. Ubp3 has several known functions, and genetic interaction data presented here suggest an additional role in transcriptional elongation. The phenotypes caused by blm3-1 and ubp3-Delta mutations are not suppressed by over-expression of BLM10, nor are they affected by deletion of BLM10. These results remove key components of the previously reported connection between Blm10/PA200-proteasome complexes and DNA repair, and they suggest a novel way to interpret sensitivity to bleomycin as resulting from defects in transcription elongation.