Nucleosomes are enriched at the boundaries of hypomethylated regions (HMRs) in mouse dermal fibroblasts and keratinocytes
- Ximiao He†1,
- Raghunath Chatterjee†1, 2,
- Desiree Tillo1,
- Andrew Smith3,
- Peter FitzGerald4 and
- Charles Vinson1Email author
© He et al.; licensee BioMed Central Ltd. 2014
Received: 6 August 2014
Accepted: 4 November 2014
Published: 2 December 2014
The interplay between epigenetic modifications and chromatin structure are integral to our understanding of genome function. Methylation of cytosine (5mC) at CG dinucleotides, traditionally associated with transcriptional repression, is the most highly studied chemical modification of DNA, occurring at over 70% of all CG dinucleotides in the genome. Hypomethylated regions (HMRs) often occur in CG islands (CGIs), however, they also occur outside of CGIs and function as cell-type specific enhancers. During the process of differentiation, reorganization of chromatin and nucleosome arrangement at regulatory regions is thought to occur in order for the establishment of cell-type specific transcriptional programs. However, the specifics regarding the organization of nucleosomes at HMRs and the potential mechanisms regulating nucleosome occupancy in these regions are unknown. Here, we have investigated nucleosome organization around hypomethylated regions (HMRs) identified in two mouse primary cells.
Microccocal nuclease (MNase) digested mononucleosomes from primary cultures of new-born female mouse dermal fibroblasts and keratinocytes were mapped and compared to the HMRs obtained from single base-pair resolution methylomes. In both cell types, we find that nucleosomes are enriched at HMR boundaries. In contrast to the nucleosomes found at boundaries of HMRs in CGIs, HMRs outside of CGIs are calculated to be preferentially bound by nucleosomes, with phased nucleosomes propagating into the methylated region. Nucleosomes are enriched at the tissue-specific HMRs (TS-HMR) boundaries in both cell types suggesting that nucleosome organization surrounding HMR boundaries is independent of methylation status. In addition, we find potential transcription factor (TF) binding sites (E-box motifs) enriched in non-CGI TS-HMR boundaries.
Our results show that intrinsic nucleosome occupancy score (INOS) positively correlate with the nucleosome organization surrounding non-CGI TS-HMRs, suggesting that DNA sequence plays a role in the establishment of HMRs in the genome. Since nucleosomes impact all processes involving the genome, our results provide a link between epigenetic modifications, chromatin structure, and regulatory function.
KeywordsCG methylation Hypomethylated regions HMR Nucleosomes Epigenomics Keratinocytes Fibroblasts
Covalent modification of different bases of DNA occurs throughout genomes [1, 2]. In mammals, the typical DNA modification is methylation of cytosine in the CG dinucleotide with over 70% of CG dinucleotides being methylated [3–5]. Several single nucleotide resolution methylation maps show that unmethylated cytosines occur in clusters representing 2% to 3% of the genome, mainly in the CG rich regions termed CG islands (CGIs), which encompass 1% of the genome. Approximately 70% of promoters contain a CGI, and these promoters tend to regulate housekeeping genes , and the hypomethylation of these regions is critical for cellular function [4, 7, 8]. However, a number of hypomethylated regions (HMRs) exist outside of CGIs (non-CGI HMRs) and promoters, and these tend to vary across cell types and tissues [4, 7–15], suggestive of a possible regulatory role for non-CGIs as enhancer elements. Indeed, non-CGI HMRs have been shown to be enriched for transcription factor binding sites (TFBSs) , and are associated with expression of nearby tissue-specific genes [4, 8, 16, 17], indicating that hypomethylation of non-CGI regions is a hallmark of regulatory function.
In addition to the methylation status of regulatory elements, the primary organizational unit of chromatin, the nucleosome, is an important factor impacting the regulatory capacity of the genome. Nucleosomes restrict access to factors requiring the DNA template and nucleosome loss, depletion, or rearrangement is indicative of transcriptional activity in regulatory regions including promoters and enhancers across species [18, 19]. Moreover, the nucleosome itself is an important factor signaling regulatory activity. For example, nucleosomes harboring specific post-translational modifications have been shown to associate with active chromatin (for example, monomethylation of lysine 4 of histone H3 is enriched in nucleosomes flanking active or poised enhancers ). Nucleosomes have also been implicated in the targeting of DNA methylation, as they have also been shown to serve as tethering sites for DNA demethylases that, in turn, contribute to the formation of repressive chromatin states [21, 22].
Several studies have begun to investigate the relationship between DNA methylation and nucleosome organization. Many studies focus on the effects of DNA methylation on nucleosome stability. It has been demonstrated that CG methylation is reduced by the presence of nucleosomes [23–27], however, methylation is enriched within nucleosome core DNA in vivo. Thus, the role of nucleosome positioning on DNA methylation as well as the effects of methylation on nucleosome positioning are not resolved, and it has been suggested that both can influence each other . Recent studies focusing on specific regulatory regions (promoters and distal enhancers) indicate that nucleosome reorganization and depletion generally accompanies demethylation of regulatory elements leading to activation of these regions in vivo[29–31]. It has recently been shown that nucleosomes tend to be highly organized over regions that are differentially methylated between tissues even when these regions are not active .
Here, we have investigated nucleosome organization at the boundaries of HMRs where the DNA methylation transition occurs. To this end, we have generated genome-wide nucleosome maps produced by MNase digestion of nucleosomal DNA obtained from primary tissues from mouse fibroblasts and keratinocytes, followed by high-throughput sequencing. We next compared the nucleosome organization surrounding HMRs that we have recently identified in both cell types [5, 32] and found that nucleosomes are enriched at the HMR boundaries. We find that nucleosome organization at non-CGI HMR boundaries, which tend to be tissue-specific, is independent of methylation status. In addition, in contrast to HMRs in CGIs, nucleosomes at the boundaries of non-CGI HMRs are predicted by a model of intrinsic nucleosome occupancy , suggesting that nucleosomes are localized at their preferred sites, implicating a role for DNA sequence in demarcating these putative regulatory regions genome-wide.
Genome-wide nucleosome maps of mouse dermal fibroblasts and keratinocytes
Micrococcal nuclease (MNase)-digested nucleosomal DNA from primary cultures of new-born female mouse fibroblasts and keratinocytes was sequenced using an Illumina HiSeq sequencer [34–38]. The resulting reads, mapped to the mouse genome, allowed the generation of map of nucleosome occupancy at high (27-35X) coverage for each cell type (Additional file 1: Table S1, Additional file 2: Figure S1).
As validation of the quality of these data, we assessed the nucleosome occupancy surrounding the transcription start site (TSS). We subdivided mouse promoters based on the presence of CGIs (+/-CGI) and their methylation status [5, 32]. We find that unmethylated promoters (+/-CGI) in both tissues are characterized by a nucleosome-depleted region upstream of the TSS, with phased nucleosomes (165 base pairs (bps) periodicity) occurring towards the body of the gene (Additional file 2: Figure S2). The degree of nucleosome depletion upstream of the TSS as well as the strength of the phasing is positively correlated with transcription, as previously observed in yeast [38, 39] and vertebrates [40–42]. The next largest group, are promoters that are methylated but do not contain a CGI (-HMR/-CGI: 8,803/28,283). In contrast to the previous groups, promoters of the most highly expressed genes in this class do not have a nucleosome depleted region or the phased nucleosome pattern similar to Saga-containing promoters in yeast  (Additional file 2: Figure S2).
Nucleosomes localize at boundaries of hypomethylated regions of keratinocytes and fibroblasts
Since a substantial fraction of HMR regions found in our methylome maps do not overlap a CGI (72.5% of all fibroblast HMRs and 81.2% of all identified keratinocyte HMRs), we next divided the HMRs into two groups: those that overlap with CGIs (Figure 1b, e, h) and those that do not (Figure 1c, f, i). For fibroblast HMRs overlapping CGIs, nucleosomes localize to the unmethylated CG boundary with phased nucleosomes extending into the methylated region, with a nucleosome depleted region in the middle of the HMR (Figure 1e). This nucleosome depleted region is at the TSS of expressed genes in CGIs (Additional file 2: Figure S2). Indeed, 78% (10,529/13,520) (Additional file 2: Figure S6a) of fibroblast HMRs within CGIs contain TSSs and the observed nucleosome depletion is presumably caused by RNA Pol II and associated factors that preferentially associate with CGI promoters (Additional file 2: Figure S2) [42, 44–46]. The 13,460 keratinocyte HMRs in CGIs are primarily promoters with nucleosomes at the boundary (Additional file 2: Figure S6c), however, the interior of these HMRs are more occupied by nucleosomes than the corresponding group in fibroblasts (Figure 1e, h), potentially reflecting the changes observed in the terminal stages of epidermal differentiation [47, 48].
Most HMRs are not in CGIs and they have a similar nucleosome pattern at the boundaries (Figure 1c, f, i). The 35,713 fibroblast and 58,035 keratinocyte HMRs without CGIs have nucleosomes at the boundary with phased nucleosomes spreading into the adjacent methylated region. However, in contrast to the HMRs within CGIs, the nucleosomes at the boundary of non-CGI HMRs are predictable by a model of intrinsic nucleosome sequence preference (intrinsic nucleosome occupancy scores (INOS))  suggesting that the localization of nucleosomes at these boundaries might be driven by DNA sequence (Figure 1f).
Nucleosome localization at non-CGI HMR boundaries does not depend on methylation status
Nucleosome organization at extended HMRs
Sequence features enriched at HMR boundaries
Our findings identify nucleosomes at boundaries of HMRs. However, the area under the ROC curve (AUC) score assessing the ability of a single CG dinucleotide to predict a nucleosome at the boundary of a HMR is modest (AUC =0.52, Additional file 2: Figure S9a). In contrast, the INOS calculation is more robust at predicting if a nucleosome will localize to these regions (AUC =0.79, Additional file 2: Figure S9a). Moreover, the predictive power of INOS is higher for the nucleosomes that are most highly enriched at HMR boundaries (that is, the top 20% of nucleosome peaks at boundaries, with an AUC =0.83 for INOS (AUC =0.81 for all the fibroblast-specific HMR boundaries), Additional file 2: Figure S9b), providing evidence that the broader sequence contexts in which these unmethylated CGs occur is important for the enrichment of nucleosomes at HMR boundaries.
We have compared genome-wide nucleosome maps in mouse fibroblasts and keratinocytes with methylome data and have found that nucleosomes are enriched at the boundaries of HMRs. Furthermore, the methylation status of the HMR does not affect nucleosome localization at the boundary. The localization of nucleosomes at the boundaries of HMRs in CGIs is not predicted by INOS, indicating that additional biochemical forces are organizing HMR boundaries. Promoters in CGIs have a similar nucleosome organization to yeast, with a nucleosome-depleted region at the TSS and phased nucleosomes extending into the gene body . However, the biochemical mechanisms that produce these positioned nucleosomes are different. In yeast, the nucleosome depleted promoter is AT rich and nucleosomes do not bind these sequences well. In contrast, mammalian promoters tend to be GC-rich and nucleosome depleted even though nucleosomes preferentially bind these sequences [45, 46]. Potentially, the DNA sequences that occur in CGIs  recruit remodelers  that are critical for the displacement of nucleosomes from favored binding positions. More impressively, in the case of HMRs outside of CGIs, which tend to be tissue-specific, we show that INOS explains a substantial fraction (AUC =0.81) of the nucleosome localization at boundaries, providing evidence that DNA sequence demarcates the boundaries of TS-HMRs in the genome. Sequence preferences of nucleosomes also play a role in the definition of the boundaries of extended HMRs, however, the exact mechanisms that regulate HMR length between tissue types warrant further investigation.
E-box motifs are enriched at boundaries of TS-HMRs in both cell types, particularly for HMRs with a nucleosome at the boundary. While some transcription factors, such as nuclear receptor proteins can bind nucleosomal DNA, B-HLH proteins cannot . Nucleosome enrichment within HMR boundaries confirms and extends previous observations regarding the encoding of nucleosome occupancy over regulatory regions [45, 46]. Since many enhancers tend to be cell-type specific, high nucleosome occupancy of TFBS can restrict their utilization to a certain cell type. In addition, nucleosomes can reinforce and promote cooperative interactions between TFs in displacing nucleosomes from their preferred sites, providing higher specificity in gene regulation [53, 54]. The E-box motif is a sequence that nucleosome preferentially binds suggesting its presence at the HMR boundary is not to be a TFBS but a nucleosome localization site. Alternatively, the E-Box may function as both, sometimes being bound by a nucleosome and other times being bound by a B-HLH protein. Whether or not HLH proteins do in fact bind these E-Box sequences, suggesting equilibrium between a nucleosome and HLH protein binding and potential enhancer function of TS-HMRs, however, remains to be determined.
We have mapped genome-wide nucleosome organization in two mouse primary tissue types, providing an important resource for the understanding of chromatin structure and gene regulation. Comparing these maps with HMRs obtained from single-base pair resolution methylomes has allowed the identification of the nucleosome arrangements in these regions. In particular, we find that nucleosomes are enriched at the boundaries of HMRs. Nucleosome organization at HMR boundaries is independent of methylation status. For HMRs not in CGI, boundaries are calculated to be well bound by nucleosome as occurs in vivo. Since hypomethylation of non-CGI regions is a hallmark of regulatory activity, our findings have important implications for the specification of chromatin architecture at regulatory regions in the genome.
Mouse primary keratinocytes and dermal fibroblasts
NIH research guidelines and IACUC approved animal study protocols were followed in this study. Keratinocytes and dermal fibroblasts were cultured from newborn wild type according to the protocol described previously . Primary keratinocytes were seeded at a density of one mouse epidermis per 10 cm dish or equivalent in calcium and magnesium free SMEM (GIBCO Laboratories, Grand Island, NY, USA), supplemented with 8% Chelex (Bio-Rad, Richmond, CA, USA) treated FBS (Atlanta Biologicals, Inc.) and 0.2 mM calcium (CaCl2). Dermal fibroblasts were also seeded at a density of one mouse dermis per 10-cm-dish or equivalent in DMEM/F12: GlutaMAX medium (Invitrogen) with 10% FBS.
Micrococcal nuclease (MNase) digestion and mapping of MNase-seq data
Primary cultures of female primary mouse dermal fibroblasts or keratinocytes were harvested and washed once with ice cold PBS. Resuspended cell pellet (approximately 108 cells) were resuspended in 5 mL of ice cold NP-40 lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) and incubated for 5 min in ice. Nuclei were washed with MNase digestion buffer (10 mM Tris–HCl (pH 7.4), 15 mM NaCl, 60 mM KCl) and resuspended in MNase digestion buffer containing 1 mM CaCl2. Nuclei were digested with MNase for 10 and 15 min at 37 C. The MNase digestion was stopped by putting the samples on ice and adding 100 mM EDTA and 10 mM EGTA (pH 7.5). MNase digested DNA were purified with the Qiagen PCR purification kit after digestion with protease K (Qiagen). Isolated DNA was eluted in elution buffer (Qiagen). Mnase digested DNA fragments were separated on a 2% agarose gel. DNA corresponding to the mononucleosomal bands was gel extracted and pulled from all digestion. The libraries for sequencing were prepared according to the standard protocol for the Illumina HiSeq2000 sequencing platform with 102 bp paired end reads. Paired end reads of MNase-seq data were aligned using Novoalign software (http://www.novocraft.com/) with default parameters for both primary dermal fibroblasts and keratinocytes to the mouse genome (UCSC build mm9). Reads mapping to more than one location were discarded, and data were filtered to include only those sequences that had a mate pair match within 100 to 160 bp. Counts were recorded at the midpoint of the mate pair alignment, and Gaussian smoothing was applied to yield a continuous measure of nucleosome signal across the entire genome .
TSS information was extracted from RefGene annotations downloaded from the UCSC genome browser (https://genome.ucsc.edu/) for version of mm9. For genes with identical transcript start and stop sites, only one was retained. We downloaded the CG island annotations from the UCSC genome browser for version mm9 of the mouse genome.
Female mouse primary dermal fibroblast and keratinocyte methylomes
The fibroblast methylome was produced and released by our group . Generation of the genome-wide primary female mouse keratinocyte methylation data is described in the accompanied paper . Regions of hypomethylation (hypomethylated regions (HMRs)) were defined using a two-state Hidden Markov Model (HMM) described in .
Intrinsic nucleosome occupancy scores
We predicted intrinsic nucleosome occupancy scores (INOSs) across HMRs using the Lasso linear model described in . For each HMR, we calculated the INOSs at every base pair using a sliding window of 147 bp.
Two biological replicates of MNase-seq for each primary dermal fibroblasts and keratinocytes are in the process of GEO submission. Keratinocyte methylome data have been submitted to the GEO database with accession number (GSE44918) . Two biological replicates of keratinocyte mRNA-seq data are in the process of submission to the GEO database . Fibroblast methylome and mRNA-seq data have been obtained from the GEO accession number (GSE44942) . The second biological replicate RNA-seq data of dermal fibroblasts are in the process of submission to the GEO database . All the sequencing data for fibroblasts and keratinocytes will be submitted to the GEO database with accession numbers (GSE44942 and GSE44918 respectively). The data can also be obtained from the authors upon request.
We thank Bao Tran, Jyoti Shetty, Yongmei Zhao, Shashikala Ratnayake, and Yuliya Kriga at the NCI CCR Sequencing Facility, Frederick, MD, USA for providing expert technical assistance with the Illumina next-generation sequencing. We would like to thank NIH Helix and Biowulf staff for providing us the cluster computational facility for our next generation data analysis. This study was supported by the Intramural Research Program of the NIH, Center for Cancer Research, National Cancer Institute.
This work is supported by the intramural funding of National Cancer Institute, National Institutes of Health, MD, USA. RC is supported by the intramural funding by the Indian Statistical Institute, India.
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