Nucleosome-depleted Regions Research Articles

Abstract 2990: Analysis of chromatin structure in the Myeloid-Lymphoid Leukemia gene translocation breakpoint cluster region

Abstract The Myeloid-Lymphoid Leukemia (MLL) gene fuses to >50 different loci as the result of reciprocal translocations associated with several subtypes of human acute leukemia. In every case, the fusion point in MLL is within the same 8 kilobase pair (kbp) region, called the MLL breakpoint cluster region (bcr). Despite this conserved breakpoint, no translocation mechanism has been definitively described. Recently, electrophoretic mobility shift assay (EMSA) was used to determine protein binding characteristics in the MLL bcr. Proteins were noted to bind at the extreme 5’ and 3’ boundaries of the MLL bcr, but not in an internal region near the 3’ end of the MLL bcr which includes the translocation breakpoint hot spot in infant and therapy-related acute myeloid leukemia patients. The limited mobility observed with the protein-DNA complexes indicate a large protein or multiple proteins may bind at the 5’ and 3’ ends of the MLL bcr. Binding occurred with nuclear proteins from hematopoetic cells, but not with nuclear proteins from fibroblast cells and was sequence-specific as shown by competitor assays. Analysis of the most 5’ region of the MLL bcr showed several discontinuous regions are important for protein binding. Our results suggest that specific protein-DNA interactions may define the limits of the MLL bcr, and that the previously described internal hotspot does not bind the same protein complex. Binding of proteins at the boundaries of the MLL bcr prompted a study of chromatin organization within the same regions of the MLL bcr. Our initial hypothesis was that the MLL bcr regions which bound the protein complex would be relatively nucleosome depleted. Using chromatin immunoprecipitation assay (ChIP), we consistently showed a lack of histone H3 at the extreme 5’ end of the MLL bcr in Hela cells, and in the B-cell leukemia cell line, REH, suggesting a nucleosome-depleted region. In repeated experiments the 3’ MLL boundary showed minimal histone H3 binding, suggesting this area is also relatively free of nucleosomes. The internal breakage hot spot showed prominent H3 binding consistent with the presence of nucleosomes. The chromatin study suggests that the MLL bcr boundary regions are more exposed, especially in the 5’ region. This further supports our EMSA findings, that the MLL bcr may be bordered by large DNA-protein complexes. The project described was supported by NIH Grant Number P20 RR016741 from the North Dakota INBRE Program of the National Center for Research Resource, and by a Minot State University institutional research grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2990.

Nucleosome-Depleted Regions in Cell-Cycle-Regulated Promoters Ensure Reliable Gene Expression in Every Cell Cycle

Many promoters in eukaryotes have nucleosome-depleted regions (NDRs) containing transcription factor binding sites. However, the functional significance of NDRs is not well understood. Here, we examine NDR function in two cell cycle-regulated promoters, CLN2pr and HOpr, by varying nucleosomal coverage of the binding sites of their activator, Swi4/Swi6 cell-cycle box (SCB)-binding factor (SBF), and probing the corresponding transcriptional activity in individual cells with time-lapse microscopy. Nucleosome-embedded SCBs do not significantly alter peak expression levels. Instead, they induce bimodal, "on/off" activation in individual cell cycles, which displays short-term memory, or epigenetic inheritance, from the mother cycle. In striking contrast, the same SCBs localized in NDR lead to highly reliable activation, once in every cell cycle. We further demonstrate that the high variability in Cln2p expression induced by the nucleosomal SCBs reduces cell fitness. Therefore, we propose that the NDR function in limiting stochasticity in gene expression promotes the ubiquity and conservation of promoter NDR. PAPERCLIP:

Schizosaccharomyces pombe genome-wide nucleosome mapping reveals positioning mechanisms distinct from those of Saccharomyces cerevisiae

Positioned nucleosomes limit the access of proteins to DNA and implement regulatory features encoded in eukaryotic genomes. Here we have generated the first genome-wide nucleosome positioning map for Schizosaccharomyces pombe and annotated transcription start and termination sites genome wide. Using this resource, we found surprising differences from the previously published nucleosome organization of the distantly related yeast Saccharomyces cerevisiae. DNA sequence guides nucleosome positioning differently: for example, poly(dA-dT) elements are not enriched in S. pombe nucleosome-depleted regions. Regular nucleosomal arrays emanate more asymmetrically-mainly codirectionally with transcription-from promoter nucleosome-depleted regions, but promoters harboring the histone variant H2A.Z also show regular arrays upstream of these regions. Regular nucleosome phasing in S. pombe has a very short repeat length of 154 base pairs and requires a remodeler, Mit1, that is conserved in humans but is not found in S. cerevisiae. Nucleosome positioning mechanisms are evidently not universal but evolutionarily plastic.

Chromatin structure is implicated in "late" elongation checkpoints on the U2 snRNA and beta-actin genes.

The negative elongation factor NELF is a key component of an early elongation checkpoint generally located within 100 bp of the transcription start site of protein-coding genes. Negotiation of this checkpoint and conversion to productive elongation require phosphorylation of the carboxy-terminal domain of RNA polymerase II (pol II), NELF, and DRB sensitivity-inducing factor (DSIF) by positive transcription elongation factor b (P-TEFb). P-TEFb is dispensable for transcription of the noncoding U2 snRNA genes, suggesting that a NELF-dependent checkpoint is absent. However, we find that NELF at the end of the 800-bp U2 gene transcription unit and RNA interference-mediated knockdown of NELF causes a termination defect. NELF is also associated 800 bp downstream of the transcription start site of the beta-actin gene, where a "late" P-TEFb-dependent checkpoint occurs. Interestingly, both genes have an extended nucleosome-depleted region up to the NELF-dependent control point. In both cases, transcription through this region is P-TEFb independent, implicating chromatin in the formation of the terminator/checkpoint. Furthermore, CTCF colocalizes with NELF on the U2 and beta-actin genes, raising the possibility that it helps the positioning and/or function of the NELF-dependent control point on these genes.

The Genomic Code for Nucleosome Positioning

Eukaryotic genomes encode an additional layer of genetic information that controls the organization of the genomic DNA into arrays of nucleosomes. This information is superimposed (multiplexed) on top of the regulatory and coding information that was previously understood. We have developed an ability to read this nucleosome positioning code and predict the in vivo locations of nucleosomes. In our most recent studies, we developed a "pure" statistical profile of nucleosome DNA sequence preferences by competitive reconstitution of nucleosomes onto genomic DNA in vitro, using a system comprising only purified genomic DNA and purified histones (the protein components of nucleosomes). Using this statistical profile, we find that genome's intrinsically encoded nucleosome positioning information accounts for the overwhelming majority of nucleosome‐occupied versus nucleosome‐depleted regions across the entire genome. Analyses of nucleosome occupancy in different biological conditions reveal that the condition‐specific action of gene regulatory proteins and chromatin remodeling factors lead to specific localized changes in nucleosome occupancy and positioning, leaving much of the chromatin organization unaffected. Our results suggest that genomes utilize the nucleosome positioning code to facilitate specific chromosome functions including to delineate functional versus nonfunctional binding sites for key gene regulatory proteins, and to define the next higher level of chromosome structure itself.

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol–chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization.

Nucleosome Depleted Region In Promoter Improves Robustness In Gene Expression

Nucleosomes assembled onto promoters are critical for eukaryotic transcription regulation. Recent genome wide mapping of nucleosome positioning in Saccharomyces cerevisiae revealed that many promoters have nucleosome depleted region (NDR) that contains the transcription factor (TF) binding sites (TFBS), presumably to provide access for the factors. However, the necessity and advantage of this configuration is not well understood. We perturbed the relative position between TFBS and nucleosomes on two G1/S cell-cycle regulated promoters, and used time-lapse fluorescence microscopy to probe their transcriptional activity in individual cells through multiple cell cycles. We find that although localization of TFBSs to NDR is not required for transcription activation, it significantly reduces variability in expression between cell cycles. TFBSs localized to NDR activate transcription in every cell cycle with high reliability. In contrast, when the same TFBSs are located within positioned nucleosomes, activation was bimodal: nearly fully active in some cell cycles, and essentially undetectable in others. Interestingly, this “on/off” transcription pattern displays short-term “memory”, or epigenic inheritance, from the previous mother cell cycle. Furthermore, the probability of “on/off” cycles could be affected by varying the TF concentration and nucleosome acetylation level. Our results have significant implications for the function and evolution of NDR, and for the relationship between the chromatin structure on promoters and the reliability of gene expression.

A high-resolution atlas of nucleosome occupancy in yeast

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.