Soft skills turned into hard facts: nucleosome remodelling at developmental switches.

Nucleosome remodelling factors are regulators of DNA accessibility in chromatin and lubricators of all major functions of eukaryotic genomes. Their action is transient and reversible, yet can be decisive for irreversible cell-fate decisions during development. In addition to the well-known local actions of nucleosome remodelling factors during transcription initiation, more global and fundamental roles for remodelling complexes in shaping the epigenome during development are emerging.

ISWI induces nucleosome sliding on nicked DNA.

The ATPase ISWI is the molecular motor of several remodeling factors that trigger nucleosome sliding in vitro. In search for the underlying mechanism, we found that unilateral binding of ISWI to a model nucleosome correlated with directional movement of the nucleosome toward the enzyme. It has been proposed that ISWI might loosen histone-DNA interactions through twisting DNA. However, nucleosome sliding assays on nicked DNA substrates suggest that propagation of altered twist is not involved. Surprisingly, nicks in the linker DNA in front of the nucleosome facilitate sliding. These data suggest that the rate of nucleosome sliding is limited by a conformational change other than twisting, such as the formation of a short loop, of DNA at the entry into the nucleosome.

Tramtrack69 interacts with the dMi-2 subunit of the Drosophila NuRD chromatin remodelling complex.

dMi-2, the ATPase subunit of the Drosophila nucleosome remodelling and histone deacetylation (dNuRD) complex, was identified in a two-hybrid screen as an interacting partner of the transcriptional repressor, Tramtrack69 (Ttk69). A short region of Ttk69 is sufficient to mediate this interaction. Ttk69, but not the Ttk88 isoform, co-purifies with the dNuRD complex isolated from embryo extracts. dMi-2 and Ttk69 co-immunoprecipitate from embryonic extracts, indicating that they can associate in vivo. Both dMi-2 and Ttk69 co-localize at a number of discrete sites on polytene chromosomes, showing that they bind common target loci. We also demonstrate that dMi-2 and Ttk interact genetically, indicating a functional interaction in vivo. We propose that Ttk69 represses some target genes by remodelling chromatin structure through the recruitment of the dNuRD complex.

Patterns and significance of exhaled-breath biomarkers in lung transplant recipients with acute allograft rejection.

BACKGROUND: Obliterative bronchiolitis (OB) remains one of the leading causes of death in lung transplant recipients after 2 years, and acute rejection (AR) of lung allograft is a major risk factor for OB. Treatment of AR may reduce the incidence of OB, although diagnosis of AR often requires bronchoscopic lung biopsy. In this study, we evaluated the utility of exhaled-breath biomarkers for the non-invasive diagnosis of AR. METHODS: We obtained breath samples from 44 consecutive lung transplant recipients who attended ambulatory follow-up visits for the Johns Hopkins Lung Transplant Program. Bronchoscopy within 7 days of their breath samples showed histopathology in 21 of these patients, and we included them in our analysis. We measured hydrocarbon markers of pro-oxidant events (ethane and 1-pentane), isoprene, acetone, and sulfur-containing compounds (hydrogen sulfide and carbonyl sulfide) in exhaled breath and compared their levels to the lung histopathology, graded as stable (non-rejection) or AR. None of the study subjects were diagnosed with OB or infection at the time of the clinical bronchoscopy. RESULTS: We found no significant difference in exhaled levels of hydrocarbons, acetone, or hydrogen sulfide between the stable and AR groups. However, we did find significant increase in exhaled carbonyl sulfide (COS) levels in AR subjects compared with stable subjects. We also observed a trend in 7 of 8 patients who had serial sets of breath and histopathology data that supported a role for COS as a breath biomarker of AR. CONCLUSIONS: This study demonstrated elevations in exhaled COS levels in subjects with AR compared with stable subjects, suggesting a diagnostic role for this non-invasive biomarker. Further exploration of breath analysis in lung transplant recipients is warranted to complement fiberoptic bronchoscopy and obviate the need for this procedure in some patients.

Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors.

ATP-dependent chromatin-remodeling machines of the SWI/SNF family are involved in many cellular processes in eukaryotic nuclei, such as transcription, replication, repair and recombination. Remodeling factors driven by the ATPase ISWI make up a subgroup of this family that exhibits defined mechanistic and functional characteristics. ISWI-induced nucleosome mobility endows nucleosomal arrays with dynamic properties and recent results suggest that ISWI-type remodelers have diverse functions that range from transcriptional regulation to chromatin assembly and maintenance of chromosome structure.

Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila.

Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. Recently, the methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. Here we show by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1, associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. We suggest a model in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome.

HMG-D and histone H1 interplay during chromatin assembly and early embryogenesis.

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1.

Acf1, the largest subunit of CHRAC, regulates ISWI-induced nucleosome remodelling.

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent 'nucleosome remodelling' activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone 'tail' requirements of the reaction. The Acf1-ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.

Association and spreading of the Drosophila dosage compensation complex from a discrete roX1 chromatin entry site.

In Drosophila, dosage compensation is controlled by the male-specific lethal (MSL) complex consisting of MSL proteins and roX RNAs. The MSL complex is specifically localized on the male X chromosome to increase its expression approximately 2-fold. We recently proposed a model for the targeted assembly of the MSL complex, in which initial binding occurs at approximately 35 dispersed chromatin entry sites, followed by spreading in cis into flanking regions. Here, we analyze one of the chromatin entry sites, the roX1 gene, to determine which sequences are sufficient to recruit the MSL complex. We found association and spreading of the MSL complex from roX1 transgenes in the absence of detectable roX1 RNA synthesis from the transgene. We mapped the recruitment activity to a 217 bp roX1 fragment that shows male-specific DNase hypersensitivity and can be preferentially cross-linked in vivo to the MSL complex. When inserted on autosomes, this small roX1 segment is sufficient to produce an ectopic chromatin entry site that can nucleate binding and spreading of the MSL complex hundreds of kilobases into neighboring regions.

Modifications of the histone N-terminal domains. Evidence for an "epigenetic code"?

A multicellular organism is made up of a variety of different cell types and tissues. This organization is accomplished by a well-concerted action of different regulatory molecules, which--in a very hierarchical manner--influence the expression of certain cell-specific genes. Many of those regulators are transcription factors, which directly influence the expression of the controlled gene by binding to a specific DNA sequence within its promoter or enhancer region. This binding then leads to an enhancement or a decrease in the rate of transcription of that particular gene and eventually regulates the production of the corresponding polypeptide. One major obstacle to the binding of these transcription factors is the fact that DNA is not readily accessible in the eukaryotic nucleus. It is associated with a class of very basic proteins called histones. This complex of histones and DNA is called chromatin.

Reconstitution of human beta-globin locus control region hypersensitive sites in the absence of chromatin assembly.

The human beta-globin genes are regulated by the locus control region (LCR), an element composed of multiple DNase I-hypersensitive sites (HS sites) located 5' to the genes. Various functional studies indicate that the LCR confers high-level, position-independent, and copy number-dependent expression to linked globin genes in transgenic mice. However, the structural basis for LCR function is unknown. Here we show that LCR HS sites can be reconstituted in an erythroid cell-specific manner on chromatin-assembled LCR templates in vitro. Surprisingly, HS2 and HS3 are also formed with erythroid proteins in the absence of chromatin assembly, indicating that sensitivity to nucleases is not simply a consequence of nucleosome reorganization. The generation of LCR HS sites in the absence of chromatin assembly leads to the formation of S1- and KMnO(4)-sensitive regions in HS2 and HS3. These sites are also sensitive to S1 nuclease in erythroid cells in vivo, suggesting a distorted DNA structure in the LCR core enhancer elements. Finally, we show that RNA polymerase II initiates transcription in the HS2 and HS3 core enhancer regions in vitro. Transcription in both HS2 and HS3 proceeds in a unidirectional manner. Taken together, the data suggest that erythroid proteins interact with the core enhancer elements, distort the DNA structure, and recruit polymerase II transcription complexes. These results further our understanding of the structural basis for LCR function and provide an explanation for why the LCR core regions are so extremely sensitive to nucleases in erythroid cells.

The histone H4 acetyltransferase MOF uses a C2HC zinc finger for substrate recognition.

Site-specific acetylation of histone H4 by MOF is central to establishing the hyperactive male X chromosome in Drosophila. MOF belongs to the MYST family of histone acetyltransferases (HATs) characterized by an unusual C2HC-type zinc finger close to their HAT domains. The function of these rare zinc fingers is unknown. We found that this domain is essential for HAT activity, in addition to the established catalytic domain. MOF uses its zinc finger to contact the globular part of the nucleosome as well as the histone H4 N-terminal tail substrate. Point mutations that leave the zinc-finger structure intact nevertheless abolish its interaction with the nucleosome. Our data document a novel role of the C2HC-type finger in nucleosome binding and HAT activity.

Critical role for the histone H4 N terminus in nucleosome remodeling by ISWI.

The ATPase ISWI can be considered the catalytic core of several multiprotein nucleosome remodeling machines. Alone or in the context of nucleosome remodeling factor, the chromatin accessibility complex (CHRAC), or ACF, ISWI catalyzes a number of ATP-dependent transitions of chromatin structure that are currently best explained by its ability to induce nucleosome sliding. In addition, ISWI can function as a nucleosome spacing factor during chromatin assembly, where it will trigger the ordering of newly assembled nucleosomes into regular arrays. Both nucleosome remodeling and nucleosome spacing reactions are mechanistically unexplained. As a step toward defining the interaction of ISWI with its substrate during nucleosome remodeling and chromatin assembly we generated a set of nucleosomes lacking individual histone N termini from recombinant histones. We found the conserved N termini (the N-terminal tails) of histone H4 essential to stimulate ISWI ATPase activity, in contrast to other histone tails. Remarkably, the H4 N terminus, but none of the other tails, was critical for CHRAC-induced nucleosome sliding and for the generation of regularity in nucleosomal arrays by ISWI. Direct nucleosome binding studies did not reflect a dependence on the H4 tail for ISWI-nucleosome interactions. We conclude that the H4 tail is critically required for nucleosome remodeling and spacing at a step subsequent to interaction with the substrate.

Chromodomains are protein-RNA interaction modules.

In Drosophila, compensation for the reduced dosage of genes located on the single male X chromosome involves doubling their expression in relation to their counterparts on female X chromosomes. Dosage compensation is an epigenetic process involving the specific acetylation of histone H4 at lysine 16 by the histone acetyltransferase MOF. Although MOF is expressed in both sexes, it only associates with the X chromosome in males. Its absence causes male-specific lethality. MOF is part of a chromosome-associated complex comprising male-specific lethal (MSL) proteins and at least one non-coding roX RNA. How MOF is integrated into the dosage compensation complex is unknown. Here we show that association of MOF with the male X chromosome depends on its interaction with RNA. MOF specifically binds through its chromodomain to roX2 RNA in vivo. In vitro analyses of the MOF and MSL-3 chromodomains indicate that these chromodomains may function as RNA interaction modules. Their interaction with non-coding RNA may target regulators to specific chromosomal sites.

dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties.

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines.

Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila.

Dosage compensation in Drosophila involves a 2-fold increase in transcription from the single male X relative to the two female X chromosomes. Regulation at the level of the chromosome involves alterations in chromatin organization: male X chromosomes appear decondensed and are marked by acetylation of histone H4 at lysine 16. We demonstrate that MOF, a protein required for dosage compensation with significant sequence similarity to the MYST family of acetyltransferases, is a histone acetyltransferase that acetylates chromatin specifically at histone H4 lysine 16. This acetylation relieves chromatin-mediated repression of transcription in vitro and in vivo if MOF is targeted to a promoter by fusion to a DNA-binding domain. Acetylation of chromatin by MOF, therefore, appears to be causally involved in transcriptional activation during dosage compensation.

HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins.

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Two histone fold proteins, CHRAC-14 and CHRAC-16, are developmentally regulated subunits of chromatin accessibility complex (CHRAC).

The ISWI ATPase of Drosophila is a molecular engine that can drive a range of nucleosome remodelling reactions in vitro. ISWI is important for cell viability, developmental gene expression and chromosome structure. It interacts with other proteins to form several distinct nucleosome remodelling machines. The chromatin accessibility complex (CHRAC) is a biochemical entity containing ISWI in association with several other proteins. Here we report on the identification of the two smallest CHRAC subunits, CHRAC-14 and CHRAC-16. They contain histone fold domains most closely related to those found in sequence-specific transcription factors NF-YB and NF-YC, respectively. CHRAC-14 and CHRAC-16 interact directly with each other as well as with ISWI, and are associated with functionally active CHRAC. The developmental expression profiles of both subunits suggest specialized roles in chromatin remodelling reactions in the early embryo for both histone fold subunits.