Epigenetic modifications and chromosome conformations of the beta globin locus throughout development

Abstract

Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic ε and fetal γ globin by the hESC-derived erythroblasts allows the interrogation of ε globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the β globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34(+) cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the β globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the ε globin promoter, as compared to the β or γ globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active ε globin promoter, similar to the loop that forms between the β or γ globin promoters and the LCR, in contrary to the previously proposed tracking mechanism.

Figure 1. The unique globin expression patterns and transcriptional machinery recruitment to the β globin locus of three developmentally distinct erythroid populations

(A) The CD45 and glycophorin-A expression by immature and mature erythroblasts derived from hESC, FL, or PB-CD34⁺ cells. Erythroblasts were generated from dissociated day-7 embryoid bodies prepared from hESCs, from FL mononuclear cells, or from mobilized PB CD34⁺ cells. Cells suspensions were collected between day 8 and day 14, stained with CD45-APC and glycophorin-A-PE, and enumerated using flow cytometry. Gatings were set based on isotype controls. (B) The β locus globins expression by the three erythroid populations. RNA was prepared from day-14 erythroblasts and RNase protection assay was carried out to determine the levels of ε, γ, and β globin transcripts. Occasionally, elevated levels of γ globin expression by PB CD34⁺ cells-derived erythroblasts were detected. A non-specific band slightly higher than the band for ε globin was detected in all samples. Results from RNase protection were also confirmed using RNA-sequencing (Supplementary Table 3). (C) Recruitment of RNA polymerase II to the β globin locus in undifferentiated hESCs and three erythroid populations. (D) Recruitment of TFIIB to the β globin locus. (E) Recruitment of TFIID to the β globin locus. For (C), (D), and (E), glycophorin-A⁺ FL erythroblasts were enriched using MACS prior to preparing chromatin for ChIP assay. The relative degree of enrichment for each target sequence was determined by first quantifying against a standard curve generated using purified human genomic DNA and then normalizing to the level of human GAPDH promoter present in each individual precipitation. Data shown are mean ± standard error of mean (SEM). One-way ANOVA was performed followed by Tukey’s Post Hoc Test to determine whether any two means of a specific target are statistically significantly different (p<0.05). When two bars are labeled with the same letter, it denotes that these means are not statistically significantly different.

Figure 2. Epigenetic landscapes of the β globin locus of undifferentiated hESCs and erythroblasts derived from hESC, FL, and PB

Quantification of (A) histone H3 acetylation (AcH3), (B) H3K4 trimethylation (H3K4me3) and (C) H3K27 monomethylation (H3K27me1) distribution across the β globin locus using ChIP assays. (A) and (B) were quantified by high throughput sequencing using Illumina Hi-seq 2000 (A), or Genome Analyzer IIX (B). Regions of local enrichment of short-read sequence tags mapped to the genome were identified using HotSpot algorithm. Data alignment was performed using Bowtie aligner. (C) was quantified by real time PCR. Normalization and statistical analyses were performed as described in Figure 1. An additional panel for the H3K27me1 enrichment in the promoter regions is provided to assist side-by-side comparison of the relative distribution of H3K27me1 across the ε, γ, and β globin promoters in each specific cell type.

Figure 3. Long range interaction between globin genes and the LCR

(A) Physical proximity between embryonic ε, fetal γ, or adult β globin promoters and the other genetic elements of the β globin locus as revealed by chromatin conformation capture studies. HindIII cut sites relative to various genetic elements are shown. Anchor fragments encompassing the promoters investigated (thick black lines) are shown in dark gray. Fragments analyzed are shown in light gray. There was insufficient amount of FL templates for analyzing the interactions between Gγ promoter and the HSs of the LCR (middle panel) but data from an independent experiment are included in Supplementary Figure 1. Data shown are mean ± standard error of mean (SEM). One-way ANOVA followed by Tukey’s Post Hoc Test was performed to analyze whether the means differ statistically significantly in the experiments where ε or β promoters were used as the anchor. For the experiment using fragment enclosing Gγ promoter as the anchor, Student’s t test was performed. * denotes that the frequencies found in hESC-derived erythroblasts are significantly different from both the FL- and PB-derived erythroblasts (top panel), or from the PB-derived erythroblasts (middle panel) * in the bottom panel indicates that the interaction frequencies found in the PB-derived erythroblasts are significantly different from those found in both FL- and hESC-derived erythroblasts. (B) Levels of transcripts across the β globin locus determined by RNA-sequencing. Data shown are genome browser view of RPKM normalized read counts mapped to the β globin locus. These read counts were further ln(x+1) transformed to enable viewing of low abundance transcripts. The untransformed RPKM normalized read counts are included in Supplementary Table 3.