Immunogenetics. Chromatin state dynamics during blood formation

Abstract

Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics; however, technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high-sensitivity indexing-first chromatin immunoprecipitation approach to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.

Figure 1. Indexing-first Chromatin ImmunoPrecipitation for profiling histone modifications and TF binding

(A) Schematic diagram of the iChIP approach to chromatin immuneprecipitation involving an initial chromatin barcoding step prior to ChIP with the desired antibody. (B) Normalized H3K4me3 profiles of peaks found in a 100 Kb region in the TNF locus (genes are indicated below) obtained with iChIP of decreasing amounts of bone-marrow derived dendritic cells (BMDC). Top, in red, H3K4me3 profile obtained using conventional ChIP with 20 million cells (15). Below, in shades of blue, H3K4me3 profiles obtained with iChIP. (C) Normalized profiles of PU.1, H3K4me1, H3K4me2, H3K4me3 and H3K27ac found in a 250 Kb region in the TNF locus obtained with iChIP with 10⁴ BMDC. (D) Scatter plots showing correlation between representative HK4me3 iChIP replicates. From left to right: correlation between 10⁴ cell replicates, 500 cell replicates, and a 500 compared to 10⁴ cells.

Figure 2. Chromatin dynamics in hematopoiesis

(A) Schematic diagram of the hematopoietic differentiation stages included in this study (platelets are displayed, but were not included). Dashed-arrow indicates intermediate progenitors not measured in this study. Color code: green for multipotent progenitors, orange for myeloid lineage (including the oligopotent progenitors CMP and GMP), blue for lymphoid lineage, red for erythroid lineage. (B) Clustering dendrogram of cell types based on H3K4me1 profiles (left) and RNA-seq levels (right) showing the differential association of lineage progenitors (CMP, GMP, MEP). (C) Representative examples of H3K4me1 signal (cell types labeled at left) in several loci, (from left to right): Gata 2 for progenitors, F7–F10 and S100a8 for myeloid lineage, Gypa and Cpox for erythroid lineage, Bcl11b for T cells. Displayed are normalized reads coverage in a 100 Kb region around the gene body. (D) Profiles of H3K4me1, H3K4me2, H3K4me3, H3K27ac modifications and RNA expression levels in two lineage specific gene loci: S100a8 in (myeloid) and Gypa (erythroid). Displayed are profiles for the lineage-specific cell types and multipotent progenitor cells over a 100 Kb region around the gene body. Putative lineage specific enhancers are shadowed in blue. Lineage specific genes are indicated below (black), as well as other genes in the loci (light gray).

Figure 3. Hematopoiesis progresses through gain and loss of lineage specific enhancers

(A) Heatmap showing 48,415 hematopoiesis enhancers clustered with K-means (K=9) of average reads count within H3K4me1 regions (16) annotated with the genomic sequence conservation of the enhancers, right (blue scale; (16)). Annotations for important lineage specific genes loci are shown on the left. (B) Schematic tree-view of three representative enhancer clusters; progenitors (V), myeloid (VI) and erythroid (IX). Color fill represents stages with H3K4me1 mark at the enhancer (C) Bar plot showing the number of enhancers gained (top, light gray) and lost (bottom, dark gray) during the development from HSC to mature hematopoietic cells along each lineage. (D) Bar plot showing the percent of de novo enhancers (from C) established at each developmental stage from HSC to mature hematopoietic cells. (E) Scatter plots showing H3K27ac signal versus H3K4me1 signal in both CMPs and EryA cells. Active enhancers (H3K4me1 positive, H3K27ac positive) are colored in red, poised enhancers (H3K4me1 positive, H3K27ac negative) in light gray, inactive enhancers (H3K4me1 negative, H3K27Ac negative) in blue. (F) Proportions of active (red) and poised (light gray) enhancers in each of the cell types studied.

Figure 4. Lineage-specific enhancers are associated with transcription factor cohorts

(A) ATAC-seq signal co-occurs temporally with H3K4me1 in lineage-specific enhancers. Shown are normalized profiles in 9 hematopoietic cells for ATAC-seq (Black) and H3K4me1 in erythroid-specific, Gypa (Red) and myeloid-specific, F7–10 loci (Orange). Displayed are peaks found in a 100 Kb region around the gene body. Putative enhancers (K4me1) shadowed in blue, transcriptional start site shadowed in red. Zoom in of ATAC-seq peak is shown below. (B) Scatter plot showing correlation between H3K4me1 signal and enhancer restricted ATAC-seq signal in four cell types: B cells, NK, MPP and CMPs (colors represent density or points). (C) Heatmap showing the p-values of transcription factor motif (Kolmagorov-Smirnov test) for the indicated cell-type-specific enhancers (16). Red indicates significant enrichment (p< 1e-5) of motif associated to the labeled TF, green indicates motif depletion (p<1e-5). White indicates either no significant enrichment or no RNA expression. (D) Bar plots showing gene expression profiles across the hematopoietic cell types for representative transcription factors (from C). (E) Schematic of the hematopoietic tree showing the representative transcription factors regulating the lineage enhancers at each cell type as predicated by the logistic model. (F) Schematic of establishment of lineage-specific enhancers in the myeloid lineage by a conventional mechanism (right) as well as via TF-mediated establishment of de novo enhancers (left).