Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF.

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.

Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- or C-termini with segmental isotope labelling for NMR spectroscopy. With access to these precisely-defined variants, we show that PTMs in both the N- and C-termini cause changes in the chemical shifts and conformational populations in regions distant from the PTM sites; up to 50-60 residues upstream of the PTM site. The PTMs investigated had only minor effects on binding of HMGN1 to nucleosome core particles, suggesting that they have other regulatory roles. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the role of PTMs with atomic precision, from both structural and functional perspectives.

Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly.

CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. Here we show that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that yields regularly spaced nucleosomes. The comparative analysis of CHD1 and ACF revealed that CHD1 assembles chromatin with a shorter nucleosome repeat length than ACF. In addition, ACF, but not CHD1, can assemble chromatin containing histone H1, which is involved in the formation of higher-order chromatin structure and transcriptional repression. These results suggest a role for CHD1 in the assembly of active chromatin and a function of ACF in the assembly of repressive chromatin.

Survey of healthcare experiences of Australian adults living with rare diseases.

BACKGROUND: Few studies have examined whether the healthcare needs of people living with rare diseases are being met. This study explores the experiences of Australian adults living with rare diseases in relation to diagnosis, information provision at the time of diagnosis, use of health and support services and involvement in research on their condition. METHODS: The survey respondents are self-selected from the population of Australian residents aged 18 years and over who are living with a rare disease. An online survey was implemented between July-August 2014. Purposive snowballing sampling was used. The results are reported as percentages with significant differences between sub-groups assessed using chi-squared analyses. RESULTS: Eight hundred ten responses were obtained from adults living with a rare disease. 92.1% had a confirmed diagnosis, of which 30.0% waited five or more years for a diagnosis, 66.2% had seen three or more doctors to get a diagnosis and 45.9% had received at least one incorrect diagnosis. Almost three quarters (72.1%) received no or not enough information at the time of diagnosis. In the 12 months prior to the survey, over 80% of respondents had used the services of a general practitioner and a medical specialist while around a third had been inpatients at a hospital or had visited an emergency department. Only 15.4% of respondents had ever used paediatric services, 52.8% of these had experienced problems in the transition from paediatric to adult services. Only 20.3% knew of a patient registry for their condition and 24.8% were informed of clinical trials. CONCLUSIONS: These findings suggest that not all healthcare needs of people living with rare diseases are being met. Structural changes to Australian healthcare systems may be required to improve the integration and coordination of diagnosis and care. Health professionals may need greater awareness of rare diseases to improve the diagnostic process and support to meet the information requirements of people newly diagnosed with rare diseases. Health service use is likely higher than for the general population and further epidemiological studies are needed on the impact of rare diseases on the healthcare system.

Distal regulatory elements play an important role in regulation of the human IL-5 gene.

Eosinophil infiltration of the lung is a feature of both allergic and nonallergic asthma, and IL-5 is the key cytokine regulating the production and activation of these cells. Despite many studies focusing on the IL-5 promoter in both humans and mice there is as yet no clear picture of how the IL-5 gene is regulated. The aim of this study was to determine if distal regulatory elements contribute to appropriate regulation of the human IL-5 (hIL-5) gene. Activity of the -507/+44 hIL-5 promoter was compared to expression of the endogenous IL-5 gene in PER-117 T cells. The IL-5 promoter was not sufficient to reproduce a physiological pattern of IL-5 expression. Further, functional analysis of the 5' and 3' intergenic regions revealed a number of novel regulatory elements. We have identified a conserved enhancer located approximately 6.2 kb upstream of the hIL-5 gene. This region contains two potential GATA-3-binding sites and increases expression from the hIL-5 promoter by up to ninefold.