RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. We addressed their roles in nucleosome phasing relative to transcription start sites in yeast. SWI/SNF has no effect on phasing at the global level. In contrast, RSC depletion results in global nucleosome repositioning: Both upstream and downstream nucleosomal arrays shift toward the nucleosome-depleted region (NDR), with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the -1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. Thus, RSC plays a critical role in organizing yeast chromatin. [Supplemental material is available for this article.]The basic subunit of chromatin structure, the nucleosome, has inhibitory effects on transcription, DNA replication, and repair, which are accounted for by its compact structure and innate stability. The nucleosome core is composed of two molecules from each of the four core histones, H2A, H2B, H3, and H4, forming a histone octamer, around which is wrapped ;147 bp of DNA (Luger et al. 1997). Early nuclease digestion studies of chromatin in nuclei revealed a characteristic ladder of DNA bands in a gel, indicating that nucleosomes are regularly spaced along the DNA in vivo. The nucleosomes in the cells of higher eukaryotes are generally spaced farther apart (;195 bp per nucleosome) than in yeast (;165 bp) (van Holde 1988). Authentic nucleosomes can be reconstituted in vitro using purified histones and DNA, but they are not regularly spaced. Regular spacing requires the addition of ATP-dependent chromatin remodeling complexes, such as ISW1, ISW2, or ACF (Ito et al. 1997;Tsukiyama et al. 1999) and is affected by linker histone (Blank and Becker 1995). The remodeling complexes use the free energy of ATP hydrolysis to move nucleosomes along DNA (Clapier and Cairns 2009;Hota and Bartholomew 2011;Narlikar et al. 2013).Genome-wide studies of nucleosomal DNA isolated from cells by digestion with micrococcal nuclease (MNase) and analyzed using tiling microarrays or massively parallel sequencing have revealed that most yeast genes have a nucleosome-depleted reg...

PurposeNiemann-Pick disease, type C (NPC) is a recessive, neurodegenerative, lysosomal storage disease caused by mutations in either NPC1 or NPC2. The diagnosis is difficult and frequently delayed. Ascertainment is likely incomplete due to both these factors and that the full phenotypic spectrum may not have been fully delineated. Given the recent development of a blood-based diagnostic test and development of potential therapies, it is important to understand the incidence of NPC and to define at risk patient populations.MethodWe evaluated data from four large massively parallel exome sequencing data sets. Variant sequences were identified and classified as pathogenic or non-pathogenic based on a combination of literature review and bioinformatic analysis. This methodology provided an unbiased approach to determining the allele frequency.ResultsOur data suggests an incidence rate for NPC1 and NPC2 of 1/92,104 and 1/2,858,998, respectively. However, evaluation of common NPC1 variants, suggests that there may be a late-onset NPC1phenotype with a markedly higher incidence on the order of 1/20,000-39,000.ConclusionsWe determined a combined incidence of classical NPC of 1/89,229 or 1.12 affected patients per 100,000 conceptions, but predict incomplete ascertainment of a late-onset phenotype of NPC1. This finding strongly supports the need for increased screening of potential patients.

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.