Nicotine-induced changes in nucleosome occupancy at the <i>LITAF</i>, <i>MLL3</i> and <i>DHFR</i> promoter regions.

<p>(A) Nucleosome positioning at the <i>LITAF</i> and (B) <i>MLL3</i> promoter after 10 minute nicotine exposure. Increased nucleosome occupancy in the nicotine treated cells (red line) relative to control cells (black line) is indicated by red bars. Nucleosome occupancy is enhanced, relative to control, at the regions just upstream of the TSS in both <i>LITAF</i> and <i>MLL3</i>, consistent with repressed transcription. (C) Nucleosome occupancy at <i>DHFR</i> is also enhanced at the TSS, above baseline and is enhanced upstream of the TSS, relative to the control, but not above baseline (pink bar). The 60 and 90 minute time points were similar to the 10 minute nicotine time point and therefore, were not shown. Nucleosome positions relative to the transcription start site in the drug naïve (black) and nicotine exposed (red) states are further illustrated pictorially at the bottom of the figure. Red and pink bars represent increased nicotine-induced nucleosome occupancy upstream of the TSS and are replicated on the pictoral illustrations at the bottom of the figure.</p

Cocaine-induced nucleosome repositioning.

<p>Comparative genome hybridization of mononucleosomally-protected DNA reveals a large-scale repositioning of nucleosomes following cocaine treatment. (A) The number of loci with significant changes in nucleosome positions fluctuates with the duration of exposure. Between 5 and 20 min, there is a significant increase in the number of loci with nucleosome repositioning (from 10 at 5 min to 211 at 20 min). However, by 40 min, the majority of the nucleosomes return to their basal positions such that only 8 loci continue to show nucleosome repositioning. At 60 min, 223 loci show significant repositioning of nucleosomes. (B) Number of genes showing nucleosome repositioning that was unique to each time point and common to multiple time points is shown. The changes detected at 5 and 40 min were common across the 20 and 60 minute time points. Some changes are unique to early or late time points (104 loci specific for 10 min, 116 loci for 60 min). (C) Nucleosome repositioning upstream of the transcription start site (TSS) and throughout the 5’ end of the <i>NFKBIB</i> gene following 5, 20 and 40 min of cocaine exposure. Nucleosome positions relative to the TSS and coding sequence of the <i>NFKBIB</i> gene in the drug naïve (black) and cocaine exposed (red) states are further illustrated pictorially at the bottom of the figure. Each sphere (black or red) represents a nucleosome. Nucleosome positions do not diverge noticeably from the basal positions at 5 min following the cocaine treatment. By 20 min, there is significant repositioning, such that nucleosomes are evicted upstream of the TSS. However, the nucleosomes return to the basal position by the 40 min (60 min identical to 40 min; data not shown). The x-axis represents the genomic position showing 2 kb centered on a TSS. The y-axis is the log₂ ratio of mononucleosomally-protected DNA to genomic DNA signal at each probe on the microarray.</p

Role of DNA sequence in nicotine- and cocaine-induced nucleosome repositioning.

<p>(A) DNA-directed nucleosome repositioning events following nicotine or cocaine treatment are shown for the <i>HDAC1</i> and <i>BAI1</i> genes. For both examples, nucleosomes were repositioned upstream and downstream of the TSS, according to the DNA-directed model. (B) DNA-independent nucleosome repositioning events following nicotine or cocaine treatment are shown for the <i>CXCL6</i> and <i>APC</i> genes, with significant remodeling events in the upstream regions and TSS of both genes. Nucleosome positions predicted by the DNA-directed model are shown as a blue line. Nucleosome illustrations (black—basal; red—nicotine treated) depict the changes in individual nucleosome positions, relative to the TSS and coding sequence of the gene model above.</p

Nucleosome Repositioning: A Novel Mechanism for Nicotine- and Cocaine-Induced Epigenetic Changes

<div><p>Drugs of abuse modify behavior by altering gene expression in the brain. Gene expression can be regulated by changes in DNA methylation as well as by histone modifications, which alter chromatin structure, DNA compaction and DNA accessibility. In order to better understand the molecular mechanisms directing drug-induced changes in chromatin structure, we examined DNA-nucleosome interactions within promoter regions of 858 genes in human neuroblastoma cells (SH-SY5Y) exposed to nicotine or cocaine. Widespread, drug- and time-resolved repositioning of nucleosomes was identified at the transcription start site and promoter region of multiple genes. Nicotine and cocaine produced unique and shared changes in terms of the numbers and types of genes affected, as well as repositioning of nucleosomes at sites which could increase or decrease the probability of gene expression based on DNA accessibility. Half of the drug-induced nucleosome positions approximated a theoretical model of nucleosome occupancy based on physical and chemical characteristics of the DNA sequence, whereas the basal or drug naïve positions were generally DNA sequence independent. Thus we suggest that nucleosome repositioning represents an initial dynamic genome-wide alteration of the transcriptional landscape preceding more selective downstream transcriptional reprogramming, which ultimately characterizes the cell- and tissue-specific responses to drugs of abuse.</p></div

Cocaine- and nicotine-induced nucleosome repositioning comparisons.

<p>(A) Changes in nucleosome repositioning that were unique to nicotine or cocaine, and common to both the drugs. (B-E) Nucleosome repositioning in response to nicotine or cocaine exposure is shown. (B) Nucleosome repositioning upstream of TSS, at the TSS and at the beginning of the coding sequence in the <i>CDNK1C</i> gene around the TSS was induced by nicotine and not by cocaine (nicotine-specific). Nicotine exposure positioned nucleosomes upstream of the TSS, depleted the nucleosomes at the TSS, and positioned nucleosomes at the start of the coding sequence. On the other hand, cocaine exposure for 20 min, did not produce changes in nucleosome position compared to the drug naïve state. (C) Nicotine and cocaine both induced nucleosome repositioning upstream of TSS, at the TSS and at the beginning of the coding sequence in the <i>ANGPT2</i> gene. However, 60 min cocaine-specific changes were detected at the +1 nucleosome (asterisk), just downstream of the <i>ANGPT2</i> TSS. The 60 min nicotine-induced nucleosome repositioning at <i>ANGPT2</i> is similar to the 20 min time point and therefore is not shown. (D) Changes common to nicotine (10 min) and cocaine (20 min) just upstream and downstream of the TSS of <i>FNB2</i> gene. (E) Neither nicotine (10 min) nor cocaine (20 min) produced nucleosome repositioning across the promoter and 5’ region of <i>BMP3</i>. Nucleosome positions relative to the TSS and coding sequence of each gene in the drug naïve (black) and drug exposed (red) states are further illustrated pictorially at the bottom of the figure. Each sphere (black or red) represents a nucleosome.</p

Changes in nucleosome position at the <i>EGR1</i> promoter region induced by nicotine exposure correlate with induction of the <i>EGR1</i> gene.

<p>Nucleosome positions relative to the transcription start site in the drug naïve (black) and nicotine exposed (red) states are further illustrated pictorially at the bottom of the figure. Each sphere (black or red) represents a nucleosome. The dashed vertical lines indicate loci of changes in nucleosome occupancy from the plot down to the nucleosome model. Based on the data from the ENCODE Consortium the red octagons indicate binding sites for transcription factors (TFs) known to repress <i>EGR1</i> expression whereas the green octagons represent binding sites for permissive TFs. Nucleosome positioning in the drug naïve basal state favored binding of repressive transcription factors (red octagons), such as LYF1/IKAROS, CTCF and EGR1 itself). However, nicotine exposure repositioned the nucleosomes such that binding sites for transcription factors such as FOS, SRF, JUNB, ZNF263, RELA, E2F1, TR4 (green squares) became depleted of nucleosomes consistent with a nucleosome configuration permissive for <i>EGR1</i> induction.</p

Nicotine-induced nucleosome repositioning.

<p>Comparative genome hybridization of mononucleosomally-protected DNA reveals a large-scale repositioning of nucleosomes following nicotine treatment. (A) Number of loci with significant nucleosome repositioning at 10, 60 and 90 minutes. (B) Number of genes showing nucleosome repositioning that was unique to each time point and common to multiple time points is shown. Common changes were in the majority with 426 out of 858 loci showing changes at every one of the time points analyzed. Some changes are unique to early or late time points (49 loci specific for 10 min, 10 loci for 60 min, and 39 loci for 90 min). (C) Nucleosomes are repositioned upstream of the transcription start site (TSS) and throughout the 5’ end of the <i>TP53</i> gene following 10, 60 and 90 min of nicotine exposure. Changes in nucleosome occupancy at the <i>TP53</i> promoter were observed at 10 min and persisted through the 60 and 90 min time points with only minor modifications. The x-axis represents the genomic position showing 2 kb centered on a TSS. The y-axis is the log₂ ratio of mononucleosomally-protected DNA to genomic DNA signal at each probe on the microarray. For all figures, changes in nucleosome position can be appreciated by comparing the divergence of the red and black lines from the grey line at the center of each image. The grey line represents theoretical zero likelihood of nucleosome occupancy. The black line is the basal nucleosome occupancy signal in the drug naïve state. The red line is the nucleosome occupancy signal following drug exposure. In each image, deflections of the black and red lines above the grey line indicate nucleosome occupancy, and deflections below the grey line indicate nucleosome depletion. Nucleosome positions relative to the TSS and coding sequence of the gene in the drug naïve (black) and drug exposed (red) states are further illustrated pictorially at the bottom of the figure. Each sphere (black or red) represents a nucleosome.</p