The genome-wide dynamics of the binding of Ldb1 complexes during erythroid differentiation

Abstract

One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex-in particular, the binding of the negative regulators Eto2 and Mtgr1-are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.

Figure 1.

Analysis of transcription factor-binding sites by ChIP-seq using Solexa Genome Analyzer II. (A) Description of the work flow used for transcription factor analysis. (B) Numbers of different complexes of the five measured factors, estimated using conservative criteria (empirical P-value of 0.05 on the experiment with the weakest signal). The factors were considered part of the same complex if the distance between centers of their binding peaks were within the resolution of the method (200 bp).

Figure 2.

Differences in transcription factor complex composition at selected loci. The number of overlapping sequence reads originating from the different ChIP-seq experiments were plotted relative to chromosomal position using Solex (red bars). Signals obtained from the control ChIP-seq experiment are also shown (green bars). Chromosomal positions as well as sequence conservation (mammalian cons.) are indicated above and below ChIP-seq plots, respectively. The figure shows clustering of all five transcription factors at the Gata1 hematopoietic enhancer (left panel), Ldb1 complex without Eto2 at the Cdh23 locus (middle panel), and Eto2 complex without Ldb1 at the Casp12 gene promoter (right panel).

Figure 3.

Dynamics of the Ldb1 complex components during the course of erythroid differentiation. ChIP-seq data showing binding of Ldb1, Tal1, Gata1, Eto2, and Mtgr1 to the Gypa, Epb4.2, Alas2, and Slc22a4 genes. (A–D) Binding profiles before (uninduced) and after (induced) differentiation. (E) ChIP analysis on nontransfected MEL cells showing enrichments of endogenous factors before (uninduced) and after (induced) differentiation. Data are represented as mean ± SEM. (F) Ratios of Ldb1 and Tal1 enrichments over Eto2 showing increases of Ldb1 and/or Tal1 after differentiation (ratios before differentiation are set to 1).

Figure 4.

Hierarchical clustering of normalized expression levels. The figure summarizes the average of normalized expression levels of gene-specific probes (annotated with the same gene name after normalization) of all samples under study. Genes that showed a significant difference (P-value of Student's t-test, <0.05) were selected. Genes with no annotation were removed, and the 100 most up-regulated and down-regulated genes were biclustered (euclidean distance, average linkage). The resulting heat map of absolute log2-transformed expression levels for uninduced and induced samples is shown. The color coding is a double gradient, where the high-end genes are shown in red and the low-end genes are shown in green.

Figure 5.

Bubble plot representation of transcription factor-binding sites around differentially regulated genes. The bubble plots encode up to four quantitative parameters per one ChIP signal: distance from the promoter of a gene (X-axis), log2 of fold change in expression of that gene between the induced and uninduced states (Y-axis), the intensity of the binding signal/peak (bubble diameter), and, optionally, different categorical information (bubble color) (A) Bubble plots showing peak size and distance to promoter of the closest gene and fold change of that gene between MEL-induced and uninduced cells. Only significantly differentially expressed genes are shown. There is a clear bias for the elements of the complex to associate with genes up-regulated upon induction (P ∼ 0, Wilcoxon test). (B) Plot for Gata1 split by the co-occurrence with other factors at the same location. The Ldb1 complex associates with up-regulated genes, while Gata1/Ldb1 without Tal1 and Eto2/Mtgr1 clusters close to promoters of both up-regulated and down-regulated genes. (C) Same as A, colored by the region of/around the reference gene. The first exon and first intron are marked separately from the rest of the internal introns and exons—the former since it is often part of the promoter, and the latter to show that it is a preferred binding region. (D) Same as A, with bubbles/peaks colored by the promoter type of the associated genes. The distribution shows that the non-CpG promoters of up-regulated genes have clear preference for complex binding close to the TSS, and that CpG island promoters respond to regulation from larger distances, most often from the first intron.

Figure 6.

Binding sites in large intergenic regions. The top panel shows two binding sites at ∼300 kb upstream of the Runx1 gene in a gene-poor region. The bottom panel shows two binding sites at ∼210 kb from the Klf3 gene. The binding profiles of the different transcription factors in uninduced and induced MEL cells are shown.

Figure 7.

Long-range interactions between ldb1 complex-bound regulatory regions. A selection of long-range interactions between the Ldb1 complex-bound β-major promoter and other ldb1-bound regulatory regions identified in 12.5-dpc fetal livers. Coordinates in megabases (Mb) on chromosome 7 are indicated at the top of each panel. The sum of the 3C sequence reads are indicated in blue. The HindIII fragments analyzed by 3C sequencing are indicated by light-gray bars. The sum of the ChIP-seq reads for Ldb1 are indicated in green; induced C88 MEL cells are on top, and uninduced C88 MEL cells are below. Genes are indicated by black bars, while the log fold change in gene expression between uniduced and induced C88 MEL cells is indicated in gray. (A) Long-range interactions between the β-major promoter and the β-globin LCR (mainly HS2 and HS4). The red bars indicate the approximate position of hypersensitive sites. (B,C) 3C-seq identifies the known long-range interactions between the β-major promoter and the Uros gene (B) and Kcnq1 region (more specifically, Tspan32) (C). Both genes are bound by the Ldb1 complex. (D) A previously unknown long-range interaction between the β-major promoter and the Suv420h2 gene is identified, which correlates with Ldb1 binding.