Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Abstract

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death.

Figure 1.

Global H4K16ac level in different types of blood cell. (A) Global H4 monoacetylation analyzed by HPLC and HPCE in neutrophils (CD15⁺ CD16⁺) and CD3⁺ T cells. Error bars represent standard deviation ***P-value <0.001. On the right, western blot comparing H4K16ac in CD3⁺ T cells versus neutrophils. H3 was used as loading control. The intensity (relative units) of H4K16ac bands were normalized to H3 and quantified using ImageJ software (numbers below the bands). (B) Levels of monoacetylated histone H4 analyzed by MALDI-TOF in the hematopoietic cells. The error bars indicate the maximum and minimum values obtained from six spectra per sample (technical replicates). (C) Fragment spectra showing that acetylation of the peptide 4–17 primarily occurs on K16 in the samples indicated. The left panel is a zoom of the region encompassing the y4 fragment ion and its acetylated form, while the right panel plots the region corresponding to b9 and its acetylated form. In both panels, the upper spectrum corresponds to the unmodified peptide 4–17 (positive control for y4), the others are the fragment spectra of the monoacetylated peptide 4–17 of the samples indicated. The detection of acetylated y4 but not its unmodified form, as well as the detection of only unmodified b9 indicate that it is mostly K16 that is acetylated. *: impurity.

Figure 2.

Genomic distribution of H4K16 acetylation in neutrophils and CD3⁺ T cells. (A) Bar plot showing the number of H4K16ac-enriched peaks identified by ChIP-seq in neutrophils and CD3⁺ T cells. On the right, a violin plot shows the distribution of the peak size in both cell types, and a bar plot shows the total coverage of the genome in base pairs (bp). (B) Box plot showing the genomic distribution of CpG density for the H4K16ac-enriched peaks in both cell types. Dotted red line represents average CpG density in the genome. (C) Bar plots showing the overlap between H4K16ac-enriched peaks and different genomic regions in both neutrophils and T cells. Violin plots represent a permutation-based null distribution for the specific genomic region overlap ratio for each peak data set. (D) Representative ChIP-seq tracks for H4K16ac in neutrophils and CD3⁺ T cells. Peaks showing fold change above 1.5 are underlined below the profiles. The RefSeq genes track is shown below. (E) Euler diagrams showing the overlap between peaks of neutrophils and CD3⁺ T cells, for both the whole genome and the different genomic regions. (F) ChromHMM training of a 12-chromatin states model (left panel) in neutrophils. The central panel shows the enrichment of each of the 12 chromatin states in different genomic features. The right panels show the percentage of neutrophil-specific segments for two neutrophil Blueprint samples (C0010KH2 and C0011lH2).

Figure 3.

Role of H4K16ac in peripheral white blood cells. (A) Diagram showing the timeline for tamoxifen administration in Kat8^fl/fl mice with and without the inducible ubiquitous Cre (Kat8^fl/fl (n: 5) and Kat8^fl/fl;ER-Cre (n: 8)). (B) Weight evolution of control and Kat8^−/−;ER-Cre mice after tamoxifen treatment. (C) Kaplan–Meier survival curve showing that Kat8^−/−; ER-Cre mice (red line) differs significantly from Kat8^fl/fl mice (blue line) (P-value = 0.0011, Kaplan–Meier log-rank test). (D) Representative images of spleen, thymus, and liver from Kat8^fl/fl and Kat8^−/−;ER-Cre mice showing differences in the size of the organs. Line plots showing (E) differences in total number of white blood cells and (F) percentage of lymphoid and myeloid cells between Kat8^fl/fl; ER-Cre and Kat8^−/− mice after tamoxifen treatment. (G). Genotype analysis of Kat8 locus using PCR specific primers on genomic DNA from Kat8^+/+;ER-Cre (pool, n: 3) as well as Kat8^−/−;ER-Cre (pool, n: 3) mice cells after tamoxifen treatment. The double band indicating approximately 30% excision of Kat8 was only detected in mice containing the floxed alleles after treatment with tamoxifen. On the right, RT-qPCR showing downregulation of Kat8 in the cKit⁺ progenitors. Expression levels determined by real time-qPCR are an average of triplicated measurements (+SD), normalized to transcript levels of beta Actin, and represented as a fold-change relative to the Kat8^+/+;ER-Cre control (a t-test showed significant differences; ***P < 0.001). (H) Representative immunofluorescence images showing lower levels of H4K16ac in Kat8^−/−;ER-Cre. Average H4K16ac fluorescence intensity was quantified using ZEN lite software (a t-test showed significant differences; **P < 0.01). (I) May Grünwald Giemsa stain (40×) after in vitro differentiation to neutrophils (day 7). (J) Expression of two markers (Collagenase and Gelatinase) of neutrophilic differentiation (Pham, C., Nature Reviews Immunology, 2006) (day 7). Expression levels determined by real time-qPCR are an average of triplicated measurements (+SD), normalized to transcript levels of beta Actin, and represented as a fold-change relative to the Kat8^+/+;ER-Cre control (a t-test showed significant differences; **P < 0.01, ***P < 0.001).

Figure 4.

Association between global levels of H4K16ac and apoptosis in myeloid cells. (A) Global H4 monoacetylation analyzed by HPLC and HPCE (blue bars in the upper panel), and percentage of Annexin⁺ cells (lower panel) during HL60 cell increasing confluence (black line). (B) Representative immunofluorescence images showing the changes in H4K16ac during the exponential growth of the cells at days 2 and 8. (C) Bar plots showing the effects of the treatment with SAHA in control and in vitro differentiated HL60 cells. Upper panel depicts the effects of the treatment on H4 monoacetylation levels, and lower panels, the effect on apoptosis measured by Annexin V-7AAD staining and percentage of debris from Propidium Iodide staining. A: ATRA, D: DMSO, S: SAHA. (D) RT-qPCR showing downregulation of KAT8 in transduced CD34⁺ progenitors. Expression levels determined by real time-qPCR are an average of triplicated measurements (+SD), normalized to transcript levels of GAPDH, and represented as a fold-change relative to the scramble. Below, a western blot comparing H4K16ac in shKAT8 cells versus the scramble. H3 was used as loading control. The intensity (relative units) of H4K16ac bands were normalized to H3 and quantified using ImageJ software (numbers below the bands). On the right, representative immunofluorescence images showing lower levels of H4K16ac in shKAT8 cells at day 11. Average H4K16ac fluorescence intensity was quantified using ZEN lite software (a t-test showed significant differences; **P < 0.001). (E) Bar plots showing the lower percentage of apoptosis of shKAT8 cells. Apoptosis levels were assessed by Annexin V-7AAD staining.

Figure 5.

Distribution of H4K16 acetylation at specific repetitive DNA elements in neutrophils and CD3⁺ T cells. (A) Bar plots showing the overlap between H4K16-enriched peaks and different types of DNA repeats, obtained in the analyses of unique mapped reads. Violin plots represent a permutation-based null distribution for each type of repeated DNA overlap ratio for each peak data set. Lower panels show associations between different types of repetitive DNA elements with H4K16ac peaks in neutrophils and CD3⁺ T cells, ranked by Q-value, and enrichment score (relative risk). (B) Enrichment of the 12 chromatin states described in Figure 2F in different types of DNA repeats. (C) Plot showing the coverage of repetitive elements in the regions flanking the H4K16ac enriched peaks obtained in the analyses of unique mapped reads in both cell types. (D) Stacked bar charts describing the classification of ambiguous reads for neutrophil and CD3⁺ T cell signals relative to the corresponding input, and according to the enrichment in different types of DNA repeats. Lower panels show associations between different types of repetitive DNA elements with probable H4K16ac-enriched regions obtained from ambiguous mapping in neutrophils and CD3⁺ T cells, and ranked by Q-value, and enrichment score (relative risk).

Figure 6.

Associations between H4K16ac enriched regions and DNA breakage during neutrophil apoptosis. (A) Schematic representation of the pulsed-field gel electrophoresis (PFGE) experiment to analyze the 50 kb DNA fragments from apoptotic neutrophils. (B) qPCR amplification of specific H4K16ac-enriched repeats using the 50kb DNA fragments from the PFGE. Data are an average of triplicated measurements, normalized to a H4K16ac-poor region of the albumin gene, and represented as a fold-change relative to the 50kb sonicated DNA. Intact DNA and 50kb random fragments from a pool of samples were used as positive controls. H4K16ac peaks identified by MACS2, and genome diagrams showing the relative enrichment of H4K16ac in neutrophils (blue), and CD3⁺ T cells (green) are shown on the left. Significant H4K16ac peaks showing fold change above 1.5 are indicated by a colored bar below the profile.

Figure 7.

Effect of H4K16ac enrichment at the repeated DNA fragmented early in neutrophil apoptosis. (A) Representative immunofluorescence images of nuclear distribution of H4K16ac in neutrophils and CD3⁺ T cells. On the right, line plots comparing DAPI and H4K16ac staining signals in several nuclear longitudinal positions. The red lines in the merged images indicates the position and direction of measurements. (B) Heat density scatterplots depicting the different distribution of the H4K16ac signal with respect to DAPI intensity in neutrophils (n = 72 cells from five donors) (upper panel) and CD3⁺ T cells (n = 62 cells from one donor) (lower panel) performed with 9198 and 5925 pairwise datapoints respectively. Colors represent the relative density of the number of measurements (data points) obtained across the different axes (blue – low density areas, red – high density areas). H4K16ac and DAPI intensities were positively correlated in lymphocyte cells (pcorr = 0.68), whereas neutrophils displayed a more sparse, non-correlated distribution (pcorr = 0.03) of H4K16ac-DAPI signals across the nucleus. (C) Chromatin accessibility assay in four donors showing that H4K16ac-enriched repetitive sequences in neutrophils are more associated with open chromatin than in CD3⁺ T cells. %FE: fold enrichment percentage.