Transcription factors TCF-1 and GATA3 are key factors for the epigenetic priming of early innate lymphoid progenitors toward distinct cell fates

Abstract

The differentiation of innate lymphoid cells (ILCs) from hematopoietic stem cells needs to go through several multipotent progenitor stages. However, it remains unclear whether the fates of multipotent progenitors are predefined by epigenetic states. Here, we report the identification of distinct accessible chromatin regions in all lymphoid progenitors (ALPs), EILPs, and ILC precursors (ILCPs). Single-cell MNase-seq analyses revealed that EILPs contained distinct subpopulations epigenetically primed toward either dendritic cell lineages or ILC lineages. We found that TCF-1 and GATA3 co-bound to the lineage-defining sites for ILCs (LDS-Is), whereas PU.1 binding was enriched in the LDSs for alternative dendritic cells (LDS-As). TCF-1 and GATA3 were indispensable for the epigenetic priming of LDSs at the EILP stage. Our results suggest that the multipotency of progenitor cells is defined by the existence of a heterogeneous population of cells epigenetically primed for distinct downstream lineages, which are regulated by key transcription factors.

Keywords: ChIC-seq; EILPs; GATA3; ILCPs; PU.1; TCF-1; epigenetic priming; multipotency; scMNase-seq; single-cell epigenomics.

Figure 1.. The dynamic epigenetic status of ILC precursors.

(A and B) UCSC genome browser images showing the landscapes of chromatin accessibility measured by DNase-seq and histone modifications measured by ChIC-seq (H3K4me3, H3K27ac, and H3K27me3) at the Tcf7 (A) and Gata3 (B) loci (also see in Figure S1 for Spi1 and Irf8 loci) in ALPs (black, top four tracks), EILPs (blue, middle four tracks), and ILCPs (red, bottom four tracks). Two independent experiments were performed for each assay. UCSC genome browser images showing the landscapes of chromatin accessibility and histone modifications at the Gata3 locus, as described in Panel A. (C) The dynamic changes of chromatin accessibility at DHSs in ILC precursors at promoter regions (upper panel) and distal regions (lower panel). Curved arrow in red color represents increased accessibility at these DHSs, blue curved arrow represents decrease accessibility at these DHSs, and horizontal dark blue arrow represents no significant changes of accessibility in these DHSs. Please also see Figure S1 and Table S1. (D) Identification of lineage-defining sites by genome-wide profiling of chromatin accessibility in ALP, EILP and ILCP cells. The DHSs detected from any of the three stages (ALP, EILP, ILCP) were separated into 7 subgroups according to their dynamic patterns indicated on the left side of the heatmap clusters. A(+)E(+)I(+) group indicates these DHSs are constitutive open; A(−)E(+)I(+) and A(−)E(−)I(+) groups (in red rectangle) indicate these DHSs increased accessibility from ALP to ILCP, either opening chromatin at EILP or ILCP stage, termed as lineage defining site-ILCs (LDS-I), while A(+)E(−)I(−) (in blue rectangle) and A(+)E(+)I(−) groups (in blue rectangle) represent the sites with decreased accessibility either at EILP stage or ILCP stage, termed as lineage defining site-ALP or alternative lineage (LDS-A). Please also see Figure S2A. (E) Genomic distribution of seven different DHSs groups in early precursor cells. Blue color presents gene promoter regions, and orange indicates distal regions. (F) Characterization of LDS-Is and LDS-As groups by motif analysis. The top four significantly enriched motifs and associated P values for LDS-Is and LDS-As are shown, respectively. (G) Gene ontology (GO) analysis reveals function terms enriched in genes associated with LDS-Is and LDS-As groups by functional annotation analysis.

Figure 2.. EILP and ILCP cells display distinct epigenetic heterogeneity at LDSs.

(A) Box plots show that ILCPs have higher accessibility than EILPs at LDS-Is measured by both nucleosome depletion (upper panel) and subnucleosomal particle enrichment (lower panel). EILP and ILCP cells were subject to single-cell MNase-seq analysis, which provided information on genome-wide nucleosome position and chromatin accessibility simultaneously at a single-cell resolution. Mann-Whitney U tests were performed to calculate the P values from about 331 single EILP and 251 ILCP cells. (B) EILPs and ILCPs show distinct epigenetic distribution profiles based on the distribution of chromatin accessibility at LDSs in two-dimensional map. X, Y axis represent the accessibility states at LDS-Is and LDS-As by open scores, respectively. Each blue dot represents a single EILP cell, and each red dot represents a single ILCP cell. The open score was calculated based on both nucleosome depletion and subnucleosomal particle enrichment at DHSs by the scMNase-seq data for individual single cells. (C) The distribution of open scores of key transcription factor motifs for TCF1, GATA3, LEF1, PU.1 and IRF8 binding motifs in EILPs (up panel) and ILCPs (low panel). X, Y axis represent the open scores at LDS-I and LDS-A, respectively. (D) The dynamic expression pattern of transcription factors TCF1, GATA3, LEF1, PU.1, IRF8 and ETS1 in ALPs, EILPs, and ILCPs. RPKMs (Reads per Kilo bases of mRNA per million mapped reads) were calculated using published RNA-Seq data (Harly et al., 2018). Data shown are representative of two independent experiments.

Figure 3.. TCF-1 is predominantly required for active epigenetic states at LDS-Is in EILP cells development.

(A) The BaGFoot (Bi-vAriate Genomic Footprints) plot showing the changed TFs by comparisons of DNase I footprints between ILCPs with ALPs. X axis value: differences in flanking accessibility; Y axis value: differences in footprint depth. Several factors, such as GATA, TCF, PU.1, are highlight with black circles. (B) Heat maps showing the profiling of chromatin accessibility (DNase-seq), TCF-1, GATA3, PU.1 and H3K27ac profiles (ChIC-seq) around LDS-Is and LDS-As from ALP to ILCP. (C) The chromatin accessibility changes at LDS-Is and LDS-As in Tcf47-deleted or Gata3-deleted EILPs. Red color, blue color, and gray color indicate increased accessibility, decreased accessibility, and no change of accessibility, respectively. The percentages of the sites with changed accessibility were indicated. Please also see Figure S3 and Figure S4. (D) Comparisons of H3K27ac signals at LDS-Is (upper panel) and LDS-As (lower panel) between TCF1-deficient and normal cells. Red color, blue color, and gray color indicate increased H3K27ac signals, decreased H3K27ac signals, and no change of H3K27ac signals, respectively. The percentages of changed H3K27ac peaks were indicated. (E) The UCSC genome browser snapshots at Tcf7, Gata3, Spi1 and Itgam gene loci, with gene structure marked at the bottom. The tracks included DNase-seq and H3K27ac ChIC-seq in ALPs, EILPs, ILCPs, Tcf7-deleted EILPs, Gata3-deleted EILPs, TCF-1 and GATA3 ChIC-seq in EILPs, ILCPs, PU.1 ChIC-seq in ALPs, EILPs. Several genomic loci were highlighted with red or bule rectangles.

Figure 4.. TCF1 is required for the epigenetic priming of LDS-Is for ILCP cells.

(A) Wild type EILPs and Tcf7-deletion EILPs show distinct distribution of epigenetic profiles at LDSs in two-dimensional map. X, Y axis represent the accessibility states at LDS-Is and LDS-As by open scores, respectively. Each blue dot represents a single wild type EILP cell, and each yellow dot represents a single Tcf7-deletion EILP cell. The open score was calculated based on both nucleosome depletion and subnucleosomal particle enrichment at DHSs by the scMNase-seq data for individual single cells. EILP1 indicates cells with a preferential open score at LDS-As; EILPs indicates cells with a preferential open score at LDS-Is. (B) The distribution of each TCF-1 deficient EILP single cell in the 2-dimentional map, based on the open scores of key transcription factor motifs for TCF-1, GATA3, LEF1, PU.1, IRF8 in each single cell, measured by scMNase-seq. (C) Plot of open score for TF binding motifs at DHSs in EILP1 (y-axis) versus EILP2 (x-axis) cells. EILP1- and EILP2-selective motifs were marked in red and green separately. Please also see Table S2. (D) The differences in open scores for the motifs of TCF-1, GATA3, LEF1, PU.1 and IRF8 in EILP1, EILP2, and Tcf7-KO EILPs. P values were calculated by performing Mann-Whitney U tests. (E) Recapturing the published tSNE plot, which shows different cell clusters in ILC progenitors (Harly, et al., 2019). Different clusters of cells are displayed with differentially colored dots. The direction of the development progression among different clusters are indicated by arrows in the schematic diagram. (F) The differences of chromatin accessibility inferred from the scRNA-seq data at LDS-As and LDS-Is across different clusters in panel E. (G) LDS-As have significantly higher proportion overlapping with DC-specific DHSs than LDS-Is and all DHSs do. (H) The difference in open scores for ILCP-specific DHSs (left panel) and DC-specific DHSs (right panel) in ILCPs, WT EILP1, WT EILP2 and Tcf7-KO EILP at single cell level. P values were calculated by performing Mann-Whitney U tests.