The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

Given our previous demonstration that RBPJ binds a methylated repressor element and regulates smooth muscle cell (SMC)-specific gene expression, we used genome-wide approaches to identify RBPJ binding regions in human aortic SMC and to assess RBPJ\'s effects on chromatin structure and gene expression. RBPJ bound to consensus cis elements, but also to TCmGGGA sequences within Alu repeats that were less transcriptionally active as assessed by DNAse hypersensitivity, H3K9 acetylation, and Notch3 and RNA Pol II binding. Interestingly, RBPJ binding was frequently detected at the borders of open chromatin, and a large fraction of genes induced or repressed by RBPJ depletion were associated with this cluster of RBPJ binding sites. RBPJ binding dramatically co-localized with serum response factor (SRF) and RNA seq experiments in RBPJ- and SRF-depleted SMC demonstrated that these factors interact functionally to regulate the contraction and inflammatory gene programs that help define SMC phenotype. Finally, we showed that RBPJ bound preferentially to phased nucleosomes independent of active chromatin marks and to cis elements positioned at the beginning and middle of the nucleosome dyad. These novel findings add important insight into RBPJ\'s role in chromatin structure and gene expression in SMC.

The nervous system may include more than 100 residue-specific posttranslational modifications of histones forming the nucleosome core that are often regulated in cell-type-specific manner. On a genome-wide scale, some of the histone posttranslational modification landscapes show significant overlap with the genetic risk architecture for several psychiatric disorders, fueling PsychENCODE and other large-scale efforts to comprehensively map neuronal and nonneuronal epigenomes in hundreds of specimens. However, practical guidelines for efficient generation of histone chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) datasets from postmortem brains are needed.
Protocols and quality controls are given for the following: 1) extraction, purification, and NeuN neuronal marker immunotagging of nuclei from adult human cerebral cortex; 2) fluorescence-activated nuclei sorting; 3) preparation of chromatin by micrococcal nuclease digest; 4) ChIP for open chromatin-associated histone methylation and acetylation; and 5) generation and sequencing of ChIP-seq libraries.
We present a ChIP-seq pipeline for epigenome mapping in the neuronal and nonneuronal nuclei from the postmortem brain. This includes a stepwise system of quality controls and user-friendly data presentation platforms.
Our practical guidelines will be useful for projects aimed at histone posttranslational modification mapping in chromatin extracted from hundreds of postmortem brain samples in cell-type-specific manner.

A method for the identification and characterization of protein-DNA interactions is presented. We have developed an approach for finding unknown multiple patterns that occur imperfectly in a set of several sequences. The pattern may contain letters from the nucleotide alphabet (A, C, G and T) including ambiguous characters (A/C, A/G, A/T; A/C/G, etc.). This method reveals weak DNA signals on an unaligned set of DNA fragments known to be functionally related and assumes no prior information on the sequences\' alignment. It determines the locations of the signals from only the information intrinsic to the sequences themselves. We have applied this method to analyze the binding sites of cAMP receptor protein (CRP). The consensus based on these data are discussed and a comparison of the consensus with the crystal structure of CAP-DNA complex is presented. We further show that in a mixture of DNA sequences, containing binding sites for two different proteins, both classes of binding sites can be discovered simultaneously by this method. The DNA sequences of nucleosome cores from chicken erythrocyte and a set of the other known nucleosomal sequences show existence of symmetrical features in nucleosome-binding DNA sequences. We also show multi-alphabet patterns that can play a role in the phasing signal on the nucleosome DNA molecule and have compared the results with existing models of nucleosome positioning.