- Open Access
Contribution of H3K4 demethylase KDM5B to nucleosome organization in embryonic stem cells revealed by micrococcal nuclease sequencing
© The Author(s) 2019
- Received: 24 January 2019
- Accepted: 26 March 2019
- Published: 2 April 2019
Positioning of nucleosomes along DNA is an integral regulator of chromatin accessibility and gene expression in diverse cell types. However, the precise nature of how histone demethylases including the histone 3 lysine 4 (H3K4) demethylase, KDM5B, impacts nucleosome positioning around transcriptional start sites (TSS) of active genes is poorly understood.
Here, we report that KDM5B is a critical regulator of nucleosome positioning in embryonic stem (ES) cells. Micrococcal nuclease sequencing (MNase-Seq) revealed increased enrichment of nucleosomes around TSS regions and DNase I hypersensitive sites in KDM5B-depleted ES cells. Moreover, depletion of KDM5B resulted in a widespread redistribution and disorganization of nucleosomes in a sequence-dependent manner. Dysregulated nucleosome phasing was also evident in KDM5B-depleted ES cells, including asynchronous nucleosome spacing surrounding TSS regions, where nucleosome variance was positively correlated with the degree of asynchronous phasing. The redistribution of nucleosomes around TSS regions in KDM5B-depleted ES cells is correlated with dysregulated gene expression, and altered H3K4me3 and RNA polymerase II occupancy. In addition, we found that DNA shape features varied significantly at regions with shifted nucleosomes.
Altogether, our data support a role for KDM5B in regulating nucleosome positioning in ES cells.
- Embryonic stem cells
- Micrococcal nuclease
- Nucleosome positioning
Nucleosomes represent the basic repeating structural unit of chromatin [1, 2], where 146 base pairs (bp) of DNA are wrapped around an octamer of histones. Nucleosomes constitute the first level of compaction of DNA into a chromatin structure in the nucleus of eukaryotic cells, and the organization of nucleosomes influences gene activity in part by controlling chromatin accessibility. Nucleosomes are depleted at transcriptional start sites (TSS), and arrays of nucleosomes are positioned adjacent to nucleosome-depleted regions [3, 4]. Covalent posttranslational modifications of histone N-terminal tails, such as methylation, are instrumental in regulating gene expression. Trimethylation of histone 3 lysine 4 (H3K4me3) is primarily localized at TSS of active genes [5–9], where it functions in part as a target for gene activation [10–12]. Lysine demethylase 5 (KDM5) family members, such as KDM5B, remove H3K4 methylation [13, 14]. KDM5B plays fundamental roles in development [15, 16], ES cell differentiation [17–19], and trophoblast stem (TS) cell differentiation . KDM5B and H3K4me3 co-localize at promoters of active genes in ES cells, where KDM5B focuses H3K4me3 near promoters to prevent it from spreading to gene bodies , an effort that may support transcriptional consistency by resetting H3K4 methylation after the transcriptional cycle by demethylating gene body regions. KDM5B performs a similar function to focus H3K4 methylation during mouse embryonic preimplantation stage development, where KDM5B demethylates broad H3K4me3 domains to prevent an increase in lengthening of H3K4me3 domains . While these results demonstrate that KDM5B regulates H3K4 methylation at promoter and gene body regions, it is unclear how KDM5B controls nucleosome organization and chromatin accessibility in ES cells. Moreover, studies have demonstrated that nucleosome distributions are influenced by transcription initiation and elongation [22, 23], processes which are controlled in part by KDM5B [18, 24]. Therefore, to clarify the role for KDM5B in regulating nucleosome organization in ES cells, we evaluated genome-wide changes in nucleosome positioning in KDM5B-depleted and control ES cells using micrococcal nuclease sequencing (MNase-Seq). Our findings demonstrate that depletion of KDM5B leads to altered enrichment of nucleosomes around TSS regions and accessible chromatin regions (DNase I hypersensitive sites). We also demonstrate that depletion of KDM5B leads to a redistribution and disorganization of nucleosomes, in a DNA sequence-dependent manner. We also uncovered non-canonical nucleosome phasing patterns and asynchronous nucleosome spacing surrounding TSS regions in KDM5B-depleted ES cells. Alterations in nucleosome distributions are correlated with dysregulated gene expression. In addition, we demonstrate that DNA shape features are significantly different at regions with shifted nucleosomes. Our data demonstrate a novel role for KDM5B in controlling nucleosome distributions in ES cells.
Evaluation of nucleosome positioning in KDM5B-depleted ES cells
Sequence reads revealed an altered nucleosome size distribution in KDM5B-depleted ES cells (Fig. 1d). In addition, nucleosome positioning varied significantly at TSS regions between control and KDM5B-depleted ES cells (Fig. 1e). To calculate variation in nucleosome positioning, we measured the mean value of distances between + 1 or − 1 nucleosomes in shLuc and shKdm5b ES cells relative to TSS regions. Results from these analyses highlight significant variation in nucleosome positioning at + 1 and − 1 nucleosomes (Fig. 1e), where nucleosomes are phased relative to TSS regions at active genes. In addition, nucleosome variation was lower at active genes (RPKM > 1) relative to inactive genes (RPKM < 1) in KDM5B-depleted ES cells (Fig. 1f). Danpos was further used to evaluate nucleosome position shifts and occupancy changes using a uniform statistical framework . To further characterize nucleosome variation in KDM5B-depleted ES cells, we stratified nucleosomes by the degree of variation between control and KDM5B-depleted ES cells (− 200 to + 200 bp) into groups of + 1 and − 1 nucleosomes relative to TSS regions that shift upstream or downstream (Fig. 1g). We then evaluated the degree of uniformity and spacing between all nucleosomes on a genome-wide scale and observed relatively similar results for control and KDM5B knockdown ES cells (Fig. 1h, left). However, an evaluation of + 1 or − 1 nucleosome arrays relative to TSSs stratified by shift distance in KDM5B-depleted ES cells (e.g., 150–250 bp) revealed altered uniformity and spacing (Fig. 1h, middle, right). Heatmaps of nucleosome density, sorted by the distance between + 1 and + 2 nucleosomes in control ES cells, also revealed variation in spacing of the + 1 nucleosome between control and KDM5B-depleted ES cells (Fig. 1i). In addition, UCSC custom browser views confirmed variation in nucleosome positioning between control and KDM5B-depleted ES cells at representative pluripotency regulators (Fig. 1j). To investigate a relationship between KDM5B binding and nucleosome shift distance in control and KDM5B-depleted ES cells, we analyzed our KDM5B ChIP-Seq data . These results revealed that + 1 or − 1 nucleosomes bound by KDM5B are more sensitive to shifting relative to regions without KDM5B binding (Fig. 1k). For − 1 nucleosomes that shifted downstream or upstream, we observed 2.4-fold or 2.6-fold more − 1 nucleosomes, respectively, bound by KDM5B relative to unbound nucleosomes (Fig. 1k). In addition, for + 1 nucleosomes that shifted downstream or upstream, we observed 2.3-fold or 2.4-fold more nucleosomes, respectively, bound by KDM5B relative to unbound nucleosomes (Fig. 1k). These findings suggest that KDM5B binding nearby TSSs is correlated with a redistribution of nucleosomes in KDM5B-depleted ES cells.
We also evaluated nucleosome enrichment and phasing surrounding TSS regions with + 1 nucleosomes exhibiting an upstream shift (Fig. 2e, f). In this case, we observed canonical nucleosome phasing for + 1 nucleosomes with small variance (upstream shift) in control and KDM5B-depleted ES cells (Fig. 2e, f). However, KDM5B-depleted ES cells exhibited non-canonical phasing patterns surrounding TSS regions with moderate to high + 1 nucleosome variance (upstream shift). These results also demonstrate slight increases in nucleosome fragment length of + 1 and − 1 nucleosomes with high variance in KDM5B-depleted ES cells (Fig. 2d, f, Additional file 1: Fig. S2D, F). Combined, these results reveal diverse nucleosome phasing patterns in ES cells and suggest that KDM5B acts in part to regulate nucleosome phasing distributions on a genome-wide scale at distinct sets of TSSs.
Furthermore, GREAT  functional annotation showed that variant + 1 or − 1 nucleosomes are located close to TSS regions of genes associated with development, differentiation, metabolism, and signaling. Overrepresentation of these gene ontology (GO) functional annotation terms was evident by semantic analysis of gene ontology (GO) terms  (Fig. 3c, d). These findings demonstrate variant + 1 and − 1 nucleosomes are associated with distinct sets of genes.
DNA sequence features of variant nucleosomes
DNA shape features of sequences containing variant nucleosomes and altered H3K4 methylation
Association of altered nucleosome positioning with changes in H3K4me3 and gene expression in KDM5B-depleted ES cells
We also investigated whether alterations in nucleosome positioning are associated with changes in RNAPII binding . In this case, we found a negative correlation between the level of RNAPII occupancy and variance of + 1 nucleosomes that shifted upstream (Fig. 6c, d). This result could suggest that an upstream shift of + 1 nucleosomes may impede RNAPII occupancy nearby TSS regions.
To investigate a relationship between nucleosome shift distance and gene expression in control and KDM5B-depleted ES cells, we analyzed RNA-Seq data . These results revealed that 3221 genes are differentially expressed between control and KDM5B-depleted ES cells (> 1.5 fold-change, FDR < 0.001). Moreover, genes associated with shifted + 1 or − 1 nucleosomes are more sensitive to transcriptional dysregulation (Fig. 6e). These findings suggest that a redistribution of nucleosomes nearby TSSs is correlated with gene expression changes in KDM5B-depleted ES cells. We also investigated a relationship between KDM5B binding , nucleosome shift distance, and transcriptional dysregulation in KDM5B-depleted ES cells. These findings show that depletion of KDM5B is associated with alterations in nucleosome positioning and transcriptional dysregulation (Fig. 6f).
Nucleosome occupancy at pluripotency regulators in KDM5B-depleted ES cells
In this study, we utilized high-resolution MNase-Seq to investigate the role for the H3K4 demethylase, KDM5B, in regulating nucleosome positioning on a global scale in ES cells. While H3K4me3  and KDM5B  are enriched at TSSs of active genes, and nucleosomes exhibit phasing relative to TSS regions, the role for KDM5B in regulating nucleosome positioning is largely unknown. Our results presented in this study demonstrate that KDM5B is a critical regulator of nucleosome positioning in ES cells. Depletion of KDM5B resulted in a redistribution of nucleosomes around regulatory regions including promoters, and asynchronous nucleosome phasing. Variation in nucleosome positioning is context specific, where positioning depends on genomic sequence and gene regulatory features such as promoters. Features of DNA shape also contribute to differences in nucleosome positioning in KDM5B-depleted ES cells. Variant nucleosomes in KDM5B-depleted ES cells may have a preference for sequences with specific conformational properties. Context-specific preferences may be correlated with H3K4 methylation level, position relative to gene regulatory regions, gene expression level, or the transcriptional cycle. Along this line, our results show that sequences nearby variant nucleosomes and gene regulatory regions such as promoters exhibit specific conformational properties.
Our results also show that active genes have a nucleosome-depleted region (NDR) upstream of the TSS and generally lack nucleosomes between the − 1 and + 1 nucleosomes. Promoter regions may be occupied by a RNAPII complex or chromatin remodeling complex, which has been suggested previously . We also observed correlations between altered H3K4me3 levels, gene expression, and nucleosome variation. These nucleosome maps provide a useful model to investigate correlations between nucleosome positioning, H3K4 methylation, and gene expression, and for understanding heterogeneity in nucleosome spacing across different sets of genes.
There are several potential mechanisms for how KDM5B may influence nucleosome positioning. First, alterations in H3K4 methylation due to depletion of KDM5B may lead to altered accessibility. While hyperacetylation of histone tails results in modest increases in chromatin accessibility [52, 53], it is unclear whether H3K4 methylation affects nucleosome positioning in vivo directly. Changes in H3K4 methylation levels in KDM5B-depleted cells may lead to altered interactions between nucleosomes and chromatin remodeling enzymes, which position nucleosomes along DNA. Along this line, several chromatin remodelers such as SMARCA5, SMARCA1, the NuRD complex protein, SIN3A, CHD1, and CHD8 were found to interact with H3K4me3 . Chromatin remodelers may cooperate with other chromatin constituents to read histone posttranslational modifications (PTMs), such as H3K4 methylation, and remodel chromatin and nucleosome placement accordingly. In this case, spreading of H3K4 methylation from promoter to gene body regions in KDM5B-depleted ES cells may direct chromatin remodelers to pattern nucleosome placement along an asynchronous array relative to normal positioning. It is important to note that we did not observe differential expression of SWI/SNF ATP-dependent chromatin remodelers such as BRG1/SMARCA4 in KDM5B-depleted ES cells. Second, KDM5B may interact with ATP-dependent chromatin remodeling factors . In support of this possibility, KDM5A and KDM5B have been shown to interact with the NuRD complex in cancer cells [56, 57]. Third, altered transcriptional dynamics in KDM5B-depleted ES cells may lead to dysregulated nucleosome positioning. Decreased RNAPII occupancy at promoters regions in KDM5B-depleted ES cells , which are generally depleted of nucleosomes at active genes, may influence nucleosome positioning. Moreover, altered RNAPII initiation and elongation rates in KDM5B-depleted ES cells may affect nucleosome positioning. Nucleosomes can inhibit the rate of transcription by occupying regulatory sequences, and nucleosomes represent a barrier to RNAPII transcription . We posit that in this kinetic model, the rate of RNAPII promoter clearance may affect nucleosome positioning. Fourth, dynamic interactions of KDM5B with chromatin constituents may affect nucleosome positioning. In this case, loss of KDM5B binding to chromatin may lead to altered nucleosome positioning. Also, our results demonstrate that expression of other KDM5 family members was unaltered in KDM5B-depleted ES cells relative to control ES cells (Additional file 1: Fig. S1), thus arguing against the possibility that altered expression of other KDM5 family members may lead to changes in H3K4 methylation and nucleosome positioning. These scenarios present intriguing possibilities for regulation of nucleosome positioning by KDM5B.
Previously, we found that depletion of KDM5B leads to delayed ES cell differentiation and dysregulated gene expression , which is in alignment with results from several studies [15, 19, 59]. There are several possible explanations for how alterations in nucleosome positioning in KDM5B-depleted ES cells may influence differentiation. Dysregulation of nucleosome positioning in KDM5B-depleted ES cells may contribute to dysregulated gene expression, nucleosome turnover, and affect response of chromatin to environmental stimuli such as intrinsic and external signals, all of which may contribute to delayed differentiation of KDM5B-depleted ES cells.
Moreover, it is possible that other KDM5 family members may play a role in regulating nucleosome positioning in ES cells or other cells. Results from previous studies suggest that KDM5B and other KDM5 family members may perform partially overlapping functions in ES cells . Along this line, KDM5A was found to regulate ES cell differentiation , and KDM5C was shown to bind promoter and enhancer regions in ES cells . However, because depletion of KDM5A or KDM5B impairs ES cell differentiation, and H3K4me3 levels increase in KDM5A or KDM5B-depleted ES cells, it is likely that KDM5 enzymes perform distinct functions.
In summary, our results describe a novel function of KDM5B in regulating nucleosome positioning in ES cells, where KDM5B-depletion leads to a redistribution of nucleosomes nearby TSSs of active genes in ES cells. These findings also serve as a resource for modeling associations between changes in H3K4 methylation and nucleosome positioning on a global scale.
ES cell culture
Mouse R1 ES cells were cultured on irradiated MEFs in DMEM/15% FBS media containing LIF (ESGRO) and 1 µg/mL puromycin at 37 °C with 5% CO2. shLuc and shKdm5b (R1) ES cells were generated and cultured as previously described with minor modifications [17, 18, 24]. Briefly, mouse R1 ES cells were transduced with lentiviral particles encoding shRNA sequences. Twenty-four hours post-transduction, KDM5B knockdown (shKdm5b) ES cells and control Luciferase-shRNA (shLuc) ES cells were stably selected in the presence of puromycin for 48–72 h, expanded for several days (3–4 days), and subsequently harvested for MNase-Seq experiments.
For MNase-Seq experiments, ES cells were cultured on gelatin-coated dishes in ES cell media containing 1.5 µM CHIR9901 (GSK3 inhibitor) for several passages to remove feeder cells. ES cells were passed by washing with PBS, and dissociating with trypsin using serological pipettes (sc-200279, sc-200281).
MNase-Seq experiments were performed as described previously with modifications . Briefly, 15e6 mouse ES cells (R1) were harvested and chemically crosslinked with 1% formaldehyde (Sigma) for 5–10 min at 37 °C. Crosslinking was quenched by addition of 1.25 M glycine to a final concentration of 0.125 M, washed with PBS twice, and the pellet was flash frozen in liquid nitrogen. Cells were thawed on ice in PBS + 0.5% Triton X-100 (lyse buffer), and nuclei were pelleted by centrifugation at 350 g for 5 min at 4 °C. Nuclei were washed with 1 mL MNase digestion buffer (10 mM tris-HCl (pH7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine), centrifuged for 5 min at 4 °C, and re-suspended in 800 µL MNase digestion buffer. The concentration of Ca2+ was adjusted to 1 mM with 1 M CaCl2. The nuclei suspension was aliquoted into tubes containing 100 µL of serially diluted MNase (0.5 U, 0.25 U, 0.125 U, 0.06 U; 1:2–1:16 dilution). The reaction was incubated for 5 min at 37 °C, and subsequently stopped by addition of stop buffer (20 mM EDTA, 20 mM EGTA, 0.4% SDS, 0.5 mg/mL proteinase K). Next, the samples were incubated at 65 °C overnight, and DNA was extracted using phenol/chloroform and precipitated with ethanol in the presence of Glycoblue. MNase-enriched DNA was run on a 2% agarose gel, and mononucleosome-sized DNA fragments were cut and pooled; MNase-enriched DNA was subjected to end-repair using an End-It DNA End-Repair kit (Epicentre), followed by addition of a single A nucleotide, and ligation of custom Illumina adapters. PCR was performed using Phusion High Fidelity PCR master mix. MNase libraries were sequenced on Illumina HiSeq platforms according to the manufacture’s protocol. Paired-end MNase-Seq reads were mapped to the mouse genome (mm9) using bowtie2  with default settings, and redundant reads were removed from further analysis. At least two replicates were performed for the MNase-Seq analyses.
MNase-Seq read enriched regions (peaks) were further investigated using Danpos, which was used to evaluate nucleosome position shifts and occupancy changes using a uniform statistical framework . The RPBM measure (read per base per million reads) was used to quantify the density at genomic regions from MNase-Seq datasets, and the UCSC genome browser was used to visualize this data.
Nucleosome occupancy relative to TSS, DNase I hypersensitive sites, CTCF-bound regions from MNase-Seq data
MNase-Seq tag densities were calculated around TSS (Fig. 1a), DHS regions (Fig. 1b), CTCF binding sites (Fig. 1c) by normalizing tag counts to total mapped tag counts. Average profiles were subsequently plotted. The positional distributions of shLuc (control) MNase-Seq reads relative to TSS, DNase I hypersensitive sites (DHS), and CTCF are consistent with previous reports [3, 27, 37, 64].
Nucleosome fragment length
Nucleosome fragments (Fig. 1d) were identified from paired-end shLuc and shKdm5b MNase-Seq data using publically available software (https://github.com/binbinlai2012/scMNase) . Nucleosome fragments with a length of 140–180 bp were considered canonical nucleosomes.
Nucleosome variance is defined as the distance between two overlapping nucleosomes  across shLuc and shKdm5b ES cells. Nucleosome variance was calculated using publically available software (https://github.com/binbinlai2012/scMNase). Briefly, nucleosome variance was measured by averaging all variances between nucleosome pairs within regions.
Nucleosome spacing uniformness
Uniformness of nucleosome spacing was calculated using publically available software (https://github.com/binbinlai2012/scMNase).
2D nucleosome occupancy heatmaps
2D nucleosome occupancy heatmaps were generated using the R package plot2DO .
H3K4me3  and RNAPII  ChIP-Seq data were mapped to the mouse genome (mm9) using bowtie2  with default settings, and redundant reads were removed from further analysis. ChIP-Seq read enriched regions (peaks) were identified relative to control Input DNA using “Spatial Clustering for Identification of ChIP-Enriched Regions” (SICER) software  with a window size setting of 200 bps, a gap setting of 400 bps, and a FDR setting of 0.001. The RPBM measure (read per base per million reads) was used to quantify the density at genomic regions from ChIP-Seq datasets. Kolmogorov–Smirnov tests were used to obtain p-value statistics and compare densities at genomic regions.
RNA-Seq data  was mapped to the mouse genome (mm9) using bowtie2  with default settings. The RPKM measure (read per kilobases of exon model per million reads)  was used to quantify the mRNA expression level of a gene from RNA-Seq data. Differentially expressed genes were identified using edgeR [false discovery rate (FDR) < 0.001; fold-change (FC) > 1.5] .
RNA extraction and quantitative real-time PCR (Q-RT-PCR) were performed as previously described with minor modifications . Total RNA was harvested from ES cells using a Qiagen RNeasy Mini Kit and DNase treated on-column. Reverse transcription was performed using an Invitrogen Superscript III kit. Primers were designed using the Roche Universal Probe Library Assay design Center.
BLK conceived of the study, designed the experiments, analyzed the sequencing data, and drafted the manuscript. JK and IOC assisted with data analysis. All authors read and approved the final manuscript.
We thank C James Block and Runsheng He for assistance with MNase-Seq library preparation. This work utilized the Wayne State University High Performance Computing Grid for computational resources (https://www.grid.wayne.edu/).
The authors declare that they have no competing interests.
Availability of data and materials
The sequencing data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE123249.
Ethics approval and consent to participate
This work was supported by Wayne State University, Karmanos Cancer Institute, and a grant from the National Heart, Lung and Blood Institute (1K22HL126842-01A1) awarded to BLK, and a grant from the Elsa U. Pardee Foundation awarded to BLK.
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