ABSTRACT

Stem cell leukemia (Scl or Tal1) and lymphoblastic leukemia 1 (Lyl1) encode highly related members of the basic helix-loop-helix family of transcription factors that are co-expressed in the erythroid lineage. Previous studies have suggested that Scl is essential for primitive erythropoiesis. However, analysis of single-cell RNA-seq data of early embryos showed that primitive erythroid cells express both Scl and Lyl1. Therefore, to determine whether Lyl1 can function in primitive erythropoiesis, we crossed conditional Scl knockout mice with mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Embryos with 20% expression of Scl from E9.5 survived to adulthood. However, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 because of progressive loss of erythropoiesis. Gene expression profiling of DKO yolk sacs revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses in a human erythroleukemia cell line showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Lyl1 can maintain primitive erythropoiesis.

INTRODUCTION

Hematopoietic stem cells (HSCs) give rise to all mature blood cells, including red blood cells (RBCs), by a process known as definitive hematopoiesis. However, the first hematopoietic cells emerge in blood islands within the yolk sac at around embryonic day (E) 7.25 (Palis, 2014). This arrival of yolk sac-derived erythroid progenitors gives rise to a wave of maturing nucleated erythroid cells that sustain the embryo between E9.5 and E12.5. Primitive erythrocytes can be distinguished from adult RBCs in the circulation by the expression of primitive globins and enucleation (Palis, 2014). In contrast, definitive or adult erythrocytes, which express adult globins, are first detected in the fetal bloodstream at around E11.5 and become the major source of circulating RBCs beginning at E13.5 (Kingsley et al., 2004).

A number of transcription factors (TFs) are known to be essential for primitive erythropoiesis. Genetic knockout models demonstrate that the TFs stem cell leukemia (Scl, also known as Tal1) and Gata1, along with co-factors Lmo2 and Ldb1, are responsible for the initiation of primitive erythropoiesis (Dore and Crispino, 2011). These factors form a large multimeric complex in erythroid cells that activates downstream targets for erythroid cell growth and differentiation. The master regulatory role for this multimeric complex is further emphasized by the ability of Scl, Lmo2 and Gata1 to directly reprogram fibroblasts to erythroid cells that resemble primitive erythroid progenitors in the yolk sac (Capellera-Garcia et al., 2016).

Scl is a hematopoietic-specific basic helix-loop-helix (bHLH) TF that is essential for the initial specification of hematopoiesis and subsequent production of RBCs and platelets (Porcher et al., 2017). It is known to form complexes with different binding partners to regulate erythropoiesis (Tripic et al., 2009; Wadman et al., 1997). Its bHLH region is highly homologous to that of the related family member known as lymphoblastic leukemia 1 (Lyl1), which is also expressed in the erythroid lineage (Visvader et al., 1991). During hematopoietic development Scl is essential, with Scl knockout mice dying in utero because of the absence of primitive erythropoiesis and vascular defects (Robb et al., 1995; Shivdasani et al., 1995). In contrast, Lyl1 knockout mice are viable, with a mild defect in stress erythropoiesis (Capron et al., 2011). These distinct knockout phenotypes can be explained by a crucial role for Scl in hematopoietic specification that cannot be replaced by Lyl1 (Chan et al., 2007). However, once hematopoiesis is established, Lyl1 can compensate for the loss of Scl in HSCs to maintain their function. It is only with the loss of both factors that there is rapid loss of HSCs due to increased apoptosis (Souroullas et al., 2009). This functional redundancy is consistent with genome-wide chromatin immunoprecipitation (ChIP)-seq studies showing shared binding of Scl and Lyl1 along with other crucial hematopoietic TFs in HSCs (Beck et al., 2013; Chacon et al., 2014; Wilson et al., 2010).

The role for Scl in primitive erythropoiesis has been difficult to delineate because of its crucial role in hematopoietic specification. Nevertheless, two studies suggest that, unlike in adult HSCs, Lyl1 cannot adequately compensate for Scl in primitive erythropoiesis. First, deletion of Scl after the formation of hematopoiesis using Scl conditional knockout mice that express the Tie2Cre transgene revealed the presence of circulating primitive erythrocytes, but mice died at E13.5-14.5 with edema, hemorrhage and dysplastic immature primitive erythroblasts (Schlaeger et al., 2005). A second study overcame hematopoietic specification by generating Scl knock-in mice that carry a germline DNA-binding mutation, in which DNA binding is not required for specification of hematopoiesis (Kassouf et al., 2008). Here, most embryos succumbed at E14.5-17.5 with anemia and defects in erythroid maturation. However, several observations raise the possibility that Lyl1 can compensate for Scl in erythropoiesis. First, one quarter of homozygous Scl DNA-binding mutant mice survive to adulthood with a mild anemia (Kassouf et al., 2008). Second, loss of one Scl allele leads to early perinatal death of Lyl1 knockout mice (Chan et al., 2007). Finally, deletion of Scl in adult HSCs led to an acute loss of erythropoiesis, but with recovery over subsequent weeks (Hall et al., 2005).

To more clearly delineate the functional redundancy of Scl and Lyl1 in erythropoiesis, we targeted Scl in erythropoiesis using the erythroid-specific erythropoietin receptor Cre-recombinase (EpoRCre) transgenic line (Heinrich et al., 2004), which is active in yolk sac blood islands from E8.5 (Drogat et al., 2010). Using this model, we show that markedly reduced expression of Scl in committed erythroid progenitors from E8.5 was compatible with embryonic development and resulted in only a mild normocytic anemia in adult mice. However, when generated on a Lyl1 knockout background, Scl/Lyl1 double knockout embryos died after E10.5 owing to the failure of primitive erythropoiesis. Gene expression profiling and ChIP-seq suggested that this functional Scl redundancy was due to shared Lyl1 and Scl target genes, including the gene encoding the crucial factor Gata1. Thus, reduced expression of Scl revealed a previously unsuspected role for Lyl1 in primitive erythropoiesis.

RESULTS

Co-expression of Scl and Lyl1 in normal primitive progenitors and erythroid cells

We first interrogated single-cell RNA-seq data obtained from E7.75 embryos to determine whether Scl and Lyl1 were co-expressed in primitive blood progenitors and erythroid cells (Scialdone et al., 2016). Four populations of cells could be defined by Scl and Lyl1 RNA expression in both blood progenitors and primitive erythroid cells, with the majority of cells (166 of 271 cells, 61%) co-expressing Scl and Lyl1 (Fig. 1A). In these cells, there was a weak correlation between Scl and Lyl1 expression (Pearson correlation coefficient r=0.17). Smaller numbers of cells expressing only Scl (‘Scl-only’) (14%), cells expressing only Lyl1 (‘Lyl1-only’) (16%) and cells apparently expressing neither (9%) were present. Overall, read counts for cells that lacked Scl or Lyl1 transcripts were lower than for cells that expressed Scl and/or Lyl1 (Fig. S1A), suggesting that the apparent lack of expression might be explained by expression below the sensitivity of sequencing. The distribution of these sub-populations was different between blood progenitors and primitive erythroid cells, with a significant increase in the proportion of Scl-only and Lyl1-only cells in the primitive erythroid cell population (χ2=47.2, P<0.00001; Fig. 1B). Gata1 and primitive globin (Hbb-bh1) expression were not significantly different between the four Scl;Lyl1-expressing subpopulations (Fig. 1C), although gene set enrichment analysis (GSEA) of the erythroid cells that lacked both Scl and Lyl1 revealed a reduction in the expression of Gata1 targets and genes that are involved in heme metabolism (Fig. S1B). Finally, erythroid cells without demonstrable Scl and Lyl1 transcripts had a reduced expression of cell cycle-related E2F target genes (Fig. 1D and Fig. S1C). Overall, single-cell gene expression analysis demonstrated the co-expression of Scl and Lyl1 expression in the majority of blood progenitors and primitive erythroid cells. Scl;Lyl1 non-expressing cells were also identified, but these may be explained by technological factors such as low read counts for TFs that are expressed at low levels.

Fig. 1.

Scl and Lyl expression in wild-type embryonic-derived progenitors and RBCs. (A) Scl and Lyl1 expression [log2 counts per million (CPM)] in single-cell blood progenitors and primitive erythroid cells from E7.75 wild-type embryos (GSE74994). The numbers in parentheses designate the numbers of progenitor and erythroid cells in the four subpopulations, respectively: Scl+Lyl1+; Scl+Lyl1; SclLyl1+ and SclLyl1. Note the SclLyl1 population is designated by a single dot as they are overlaid. (B) Proportion of the four subpopulations in blood progenitors (Prog) and primitive erythroid cells (Ery). (C) Expression of embryonic globin (Hbb-bh1) and Gata1 in the four subpopulations shown as a box plot with median. Whiskers show maximum and minimum values. (D) GSEA analysis of differentially expressed genes between cells expressing Scl and Lyl1 and cells not expressing Scl and Lyl1 that demonstrates enrichment in E2F gene targets.

Fig. 1.

Scl and Lyl expression in wild-type embryonic-derived progenitors and RBCs. (A) Scl and Lyl1 expression [log2 counts per million (CPM)] in single-cell blood progenitors and primitive erythroid cells from E7.75 wild-type embryos (GSE74994). The numbers in parentheses designate the numbers of progenitor and erythroid cells in the four subpopulations, respectively: Scl+Lyl1+; Scl+Lyl1; SclLyl1+ and SclLyl1. Note the SclLyl1 population is designated by a single dot as they are overlaid. (B) Proportion of the four subpopulations in blood progenitors (Prog) and primitive erythroid cells (Ery). (C) Expression of embryonic globin (Hbb-bh1) and Gata1 in the four subpopulations shown as a box plot with median. Whiskers show maximum and minimum values. (D) GSEA analysis of differentially expressed genes between cells expressing Scl and Lyl1 and cells not expressing Scl and Lyl1 that demonstrates enrichment in E2F gene targets.

Markedly reduced expression of Scl during development is sufficient for survival to adulthood

To determine the role of Scl in developmental erythropoiesis, we crossed Scl conditional knockout mice (SclcKO) (Hall et al., 2003) with EpoRCre mice, which express Cre after the initiation of erythropoiesis from E8.5 (Drogat et al., 2010). Mating of EpoRCre mice with EYFP reporter mice confirmed the presence of Cre activity from the colony forming unit-erythroid stage (CFU-e) onwards (Fig. S2). Deletion of Scl from E8.5 did not lead to embryonic lethality: EpoRCreSclcKO mice (ESclcKO) were born at the expected Mendelian ratio with a mild normocytic anemia (Table 1). Analysis of whole yolk sacs from ESclcKO E9.5 embryos demonstrated a reduced but detectable (19%) expression of Scl (Fig. 2A). Expression of Scl in the absence of Lyl1 was not increased. Fluorescence-activated cell sorting (FACS) analysis of adult ESclcKO bone marrow demonstrated an increase in immature Ter119loCD71+ erythroblasts (Fig. 2B) similar to that seen in MxCreSclcKO mice (Hall et al., 2005). As expected, absence of Lyl1 had no effect on steady state bone marrow erythropoiesis (Fig. 2B). Similar to the ESclcKO E9.5 embryos, quantitative real-time PCR (qPCR) of sorted bone marrow Ter119hiCD71+ erythroblasts demonstrated a 20% residual expression of Scl mRNA in ESclcKO but no compensatory increase in Lyl1 expression (Fig. 2C). The reduced expression of Scl in ESclcKO erythroid cells had no effect on the growth of CFU-e in vitro (Fig. 2D). Taken together, these results show that the significantly reduced expression of Scl after E8.5 was compatible with embryonic survival to adulthood.

Table 1.

RBC parameters of adult mice

RBC parameters of adult mice
RBC parameters of adult mice
Fig. 2.

Erythropoiesis in single knockout mice. (A) Expression of Scl in E9.5 yolk sacs from wild-type (WT), EpoRCreSclf/f (ESclcKO) and Lyl1KO embryos. (B) Representative FACS plots of WT, ESclcKO and Lyl1KO adult bone marrow stained with Ter119 and CD71 showing gates that define pro-erythroblasts (CD71+Ter119lo), intermediate erythroblasts (CD71+Ter119+) and late erythroblasts (CD71Ter119+). (C) Expression of Scl (left panel) and Lyl1 (right panel) in FACS-isolated intermediate erythroblasts from adult mice. (D) The number of erythroid colony forming units (CFU-erythroid) per 50,000 bone marrow cells from adult WT and ESclcKO mice. Data are mean±s.e.m. of three mice for each genotype.

Fig. 2.

Erythropoiesis in single knockout mice. (A) Expression of Scl in E9.5 yolk sacs from wild-type (WT), EpoRCreSclf/f (ESclcKO) and Lyl1KO embryos. (B) Representative FACS plots of WT, ESclcKO and Lyl1KO adult bone marrow stained with Ter119 and CD71 showing gates that define pro-erythroblasts (CD71+Ter119lo), intermediate erythroblasts (CD71+Ter119+) and late erythroblasts (CD71Ter119+). (C) Expression of Scl (left panel) and Lyl1 (right panel) in FACS-isolated intermediate erythroblasts from adult mice. (D) The number of erythroid colony forming units (CFU-erythroid) per 50,000 bone marrow cells from adult WT and ESclcKO mice. Data are mean±s.e.m. of three mice for each genotype.

Lyl1 can substitute for Scl in primitive erythropoiesis

As previously described (Capron et al., 2011), mice that lack Lyl1 (Lyl1KO) had a mild RBC phenotype with macrocytosis (Table 1). We bred ESclcKO mice with Lyl1KO mice to determine whether Lyl1 was providing the redundancy in primitive erythropoiesis with reduced Scl expression. Consistent with this hypothesis, mice with the loss of both Scl and Lyl1 in erythropoiesis did not survive to weaning. Timed mating identified pale embryos from E10.5 onwards (Fig. 3A), with loss of the fetal heartbeat by E11.5 and complete absence of ESclcKO;Lyl1KO (DKO) embryos by E12.5 (Table 2). Histological sections of E10.5 DKO yolk sacs demonstrated a decrease in the number of nucleated RBCs (Fig. 3B). Benzidine staining of DKO yolk sac cells showed the progressive loss of benzidine+ cells from E10.5, with the reduced intensity of staining indicative of reduced heme (Fig. 3C,D).

Fig. 3.

Embryos lacking Lyl1 and reduced Scl die owing to failure of erythropoiesis. (A) Representative E10.5 EpoRCret/+Sclf/fLyl1k/k embryo (DKO) showing pale embryo compared with a Lyl1 heterozygous (Lyl1Het) embryo and single knockout embryos (ESclcKO and Lyl1KO). (B) Benzidine staining of yolk sac cells demonstrating the decreased number of globin-expressing cells (orange) in E10.5 DKO embryos. (C) Representative FACS profiles of wild-type, ESclcKO, Lyl1KO and DKO E10.5 yolk sacs. (D) Proportion of erythroid fractions are mean±s.e.m. of four mice for each genotype. (E) Number of benzidine+ cells per high-power field (HPF) from wild-type (WT), ESclcKO, Lyl1KO and DKO embryos from E9.5 to E12.5. Data are mean±s.e.m. of four mice for each genotype.

Fig. 3.

Embryos lacking Lyl1 and reduced Scl die owing to failure of erythropoiesis. (A) Representative E10.5 EpoRCret/+Sclf/fLyl1k/k embryo (DKO) showing pale embryo compared with a Lyl1 heterozygous (Lyl1Het) embryo and single knockout embryos (ESclcKO and Lyl1KO). (B) Benzidine staining of yolk sac cells demonstrating the decreased number of globin-expressing cells (orange) in E10.5 DKO embryos. (C) Representative FACS profiles of wild-type, ESclcKO, Lyl1KO and DKO E10.5 yolk sacs. (D) Proportion of erythroid fractions are mean±s.e.m. of four mice for each genotype. (E) Number of benzidine+ cells per high-power field (HPF) from wild-type (WT), ESclcKO, Lyl1KO and DKO embryos from E9.5 to E12.5. Data are mean±s.e.m. of four mice for each genotype.

Table 2.

Numbers of DKO embryos

Numbers of DKO embryos
Numbers of DKO embryos

To assess the erythroid compartment of the DKO embryos in more detail, E10.5 yolk sacs were digested with collagenase and single-cell suspensions generated for FACS analysis (Fig. 3E). ESclcKO erythroblasts had a mildly reduced expression of Ter119, a known transcriptional target of Scl (Kassouf et al., 2008; Lahlil et al., 2004). In contrast, DKO embryos had no detectable expression of surface Ter119. Therefore, normal expression of either Scl or Lyl1 was required to maintain primitive erythropoiesis and survival beyond E10.5.

Scl and Lyl1 regulate Gata1 and shared downstream targets in primitive erythropoiesis

To gain insight into the regulation of primitive erythropoiesis by Scl and Lyl1, we performed RNA-seq analysis on cells from E9.5 yolk sacs, a time before any significant loss of benzidine+ erythroblasts in DKO embryos (Fig. 3D). Markedly reduced, but not absent, expression of the lox-P-flanked exon 5 of Scl (10% of wild-type levels) was observed in ESclcKO yolk sacs (Fig. 4A and Fig. S3). DKO yolk sacs also had markedly reduced expression of Scl, apart from one yolk sac in which expression was only reduced by ∼50% (Fig. S3). Residual expression of Scl might be explained by either incomplete deletion in primitive erythropoiesis or expression in endothelial cells, given that the expression analysis was performed using whole yolk sacs that contain endothelial cells. As expected for a constitutive knockout, Lyl1 mRNA was essentially absent in Lyl1KO and DKO yolk sac cells (Fig. S3). Overall, the RNA-seq analysis at E9.5 confirmed the absence of Lyl1 expression and the markedly reduced, but not absent, expression of Scl exon 5 in the knockout yolk sacs.

Fig. 4.

RNA-seq analysis of E9.5 yolk sacs. (A) Expression of exon 5 of Scl in wild-type (WT), ESclcKO, Lyl1KO and DKO E9.5 yolk sacs. Data are mean±s.e.m. of three mice for each genotype.  (B) Venn diagram showing numbers of differentially expressed genes in ESclcKO, Lyl1KO and DKO E9.5 yolk sacs compared with wild-type E9.5 yolk sacs using a false discovery rate of 0.01. (C) GSEA of genes involved in extracellular matrix organization in ESclcKO cells. (D) GSEA of genes involved in heme metabolism in DKO cells. (E) Heat map showing expression of leading edge heme-related genes from panel C. (F) qPCR of Gata1 in WT, ESclcKO, Lyl1KO and DKO cells. Expression normalized for Hprt and expressed relative to WT. Data are mean±s.e.m. of three pools of yolk sacs for each genotype. *P<0.05 (Student's t-test). (G) GSEA of Gata1 target genes in DKO cells with leading edge genes listed.

Fig. 4.

RNA-seq analysis of E9.5 yolk sacs. (A) Expression of exon 5 of Scl in wild-type (WT), ESclcKO, Lyl1KO and DKO E9.5 yolk sacs. Data are mean±s.e.m. of three mice for each genotype.  (B) Venn diagram showing numbers of differentially expressed genes in ESclcKO, Lyl1KO and DKO E9.5 yolk sacs compared with wild-type E9.5 yolk sacs using a false discovery rate of 0.01. (C) GSEA of genes involved in extracellular matrix organization in ESclcKO cells. (D) GSEA of genes involved in heme metabolism in DKO cells. (E) Heat map showing expression of leading edge heme-related genes from panel C. (F) qPCR of Gata1 in WT, ESclcKO, Lyl1KO and DKO cells. Expression normalized for Hprt and expressed relative to WT. Data are mean±s.e.m. of three pools of yolk sacs for each genotype. *P<0.05 (Student's t-test). (G) GSEA of Gata1 target genes in DKO cells with leading edge genes listed.

Multi-dimensional scaling using the 500 most differentially regulated genes confirmed the clustering of samples according to genotype (Fig. S4). Using a false discovery rate of 0.01, 156 genes were differentially expressed (32 up and 124 down) in ESclcKO yolk sacs and 310 genes (101 up and 209 down) in Lyl1KO yolk sacs (Fig. 4B and Table S1). GSEA showed downregulation of collagen and extracellular matrix genes in single knockouts (Fig. 4C and Fig. S4B). In yolk sacs from DKO embryos, 568 genes were differentially expressed (168 up and 400 down), with more than 70% (401) unique to the DKO embryos (Fig. 4B). Analysis of DKO cells also revealed a reduction in extracellular matrix organization genes, which was further reduced compared with Lyl1KO but not with ESclcKO cells (Fig. S4B). Unlike single knockouts, GSEA analysis of DKO cells revealed the downregulation of genes that are involved in heme metabolism (Fig. 4D,E). Of note, there was no evidence of increased apoptosis genes in DKO cells (Fig. S4C).

Gata1 is a known target gene of Scl, with both factors participating in a multimeric complex regulating erythropoiesis (Tripic et al., 2009; Wadman et al., 1997). Gata1 expression was normal in ESclcKO cells but reduced twofold in Lyl1KO cells and, most markedly, reduced tenfold in DKO cells (Fig. 4F). Consistent with the loss of Gata1, the expression of reported targets of the SCL-GATA1 complex, including Kruppel-like factor 1 (Klf1), FOG family member 1 (Zfpm1) and transferrin receptor (Tfrc) were reduced in DKO cells (Fig. 4G). However, loss of both Scl and Lyl1 did not affect the expression of genes encoding other members of the SCL-GATA1 complex, including Tcf3 (also known as E47), Ldb1, Lmo2 and the co-repressor Cbfa2t3 (also known as Eto-2) (Fig. S5A). Of note, expression of both Gata2 and Runx1 was increased in DKO cells (Fig. S5B).

Previous studies have implicated a role for Gata1 in survival of erythroblasts (Weiss and Orkin, 1995), which might explain the loss of benzidine+ erythroblasts between E9.5 and E10.5 (Fig. 2D). However, p53 target genes were not increased in DKO cells (Fig. S6A). In contrast, and similar to wild-type E7.75 primitive erythroid cells with no detectable Scl and Lyl1 transcripts (Fig. 1D), there was a reduction in E2F target genes in DKO cells (Fig. S6B and S6C). In summary, gene expression analyses before the loss of benzidine-staining cells in E9.5 yolk sac demonstrated the reduced expression of Gata1 and its targets in DKO cells.

LYL1 binds a small subset of SCL targets

We used ChIP to identify the common binding targets of Scl and Lyl1 in erythropoiesis. None of the commercially available anti-Lyl1 antibodies were able to pull down mouse Lyl1 and therefore we used the human erythroleukemia cell line K562 for ChIP experiments to compare SCL and LYL1 DNA binding. Overall, 23,547 SCL- and 1409 LYL1-binding peaks were identified in the K562 genome (Fig. 5A and Table S2). Most strikingly, 1405 (99.7%) of the LYL1-binding sites were also bound by SCL, indicating that LYL1 almost exclusively binds a small subset of SCL targets. Importantly, overlapping binding was seen at the GATA1 gene locus (Fig. 5B) as well as at loci of other erythroid-specific TFs (KLF1 and ZFPM1), heme synthetic enzymes and RBC membrane proteins (Fig. S7A-C), and at the locus control region of the β-globin locus. The shared SCL- and LYL1-binding sites were enriched for GATA1, E-BOX, RUNX and ETS motifs (Fig. 5C). Furthermore, the majority of SCL and LYL1 shared binding sites (1157/1405, 82%) were also occupied by GATA1 with or without RUNX1 (Fig. 5D and Table S2). In summary, genome-wide ChIP-seq analysis in K562 human erythroleukemic cells shows that LYL1 binds a small subset (6%) of SCL targets that are also bound by GATA1.

Fig. 5.

LYL1 binds a small subset of SCL targets including GATA1. (A) Venn diagram showing numbers of common and unique binding peaks for SCL and LYL1 in the human erythroid leukemia cell line, K562. (B) Read peak plot for ChIP-seq reads in K562 cells displayed in the UCSC genome browser (genome.ucsc.edu) shows overlapping binding of SCL and LYL1 near the GATA1 gene. (C) De novo motif discovery of LYL1 and SCL-binding sites in K562 cells has identified significant enrichment for the GATA, E-BOX, RUNX and ETS motifs. (D) Venn diagram showing numbers of shared and unique binding peaks for SCL, LYL1, GATA1 and RUNX1 in K562 cells.

Fig. 5.

LYL1 binds a small subset of SCL targets including GATA1. (A) Venn diagram showing numbers of common and unique binding peaks for SCL and LYL1 in the human erythroid leukemia cell line, K562. (B) Read peak plot for ChIP-seq reads in K562 cells displayed in the UCSC genome browser (genome.ucsc.edu) shows overlapping binding of SCL and LYL1 near the GATA1 gene. (C) De novo motif discovery of LYL1 and SCL-binding sites in K562 cells has identified significant enrichment for the GATA, E-BOX, RUNX and ETS motifs. (D) Venn diagram showing numbers of shared and unique binding peaks for SCL, LYL1, GATA1 and RUNX1 in K562 cells.

Previous studies using mice with a mutant form of Scl that does not bind DNA (Scl DNA-binding mutant mice) have identified 594 genome regions (∼20% of all Scl-binding sites) that could bind in the absence of direct Scl binding (Kassouf et al., 2010). To test the possibility that LYL1 might be able to replace SCL at these target regions, LYL1 and SCL common binding sites in K562 cells (1405 regions) were compared with Scl-binding regions in the Scl DNA-binding mutant mice (GSE18720). These 594-mouse genome regions were mapped to 494 human-genome regions. Intersection with the 1405 SCL and LYL1 shared binding sites identified in K562 cells revealed a significant overlap of 83 common sites (Table S3, z-score 25.96). Importantly, this overlap was more significant than the intersection of the remaining 2400 (80%) SCL-dependent binding sites with the same 1405 SCL and LYL1 shared binding sites (Table S3, z-score 12.61). Pathway analysis of the 75 nearest genes showed enrichment for embryonic hematopoiesis (Table S3) that included Gata1, Runx1, Lmo2 and Gfi1. Therefore, the previously identified SCL-binding sites that are independent of direct SCL binding are enriched for LYL1-binding sites.

Scl and Lyl1 directly regulate Gata1 and its targets

To identify the functionally relevant shared Scl- and Lyl1-binding targets, we integrated ChIP-seq and RNA-seq data. We mapped the 1405 shared targets from K562 to 1403 mouse homologs (Table S2). Taken together, we found that 127 genes were differentially expressed in either single knockout or DKO mice. Of these, the majority (80 genes, 63%) were differentially expressed in the DKO mice only (Fig. 6A, Fig. S8 and Table S2). The majority of these shared SCL and LYL1 targets were repressed (59 genes, 74%) including Gata1, Klf1 and Zpfm1, whereas the remaining genes (21 genes, 26%) were upregulated and included Runx1. We confirmed these findings using Scl ChIP-seq data obtained from purified hematopoietic progenitors derived from mouse embryonic stem cells (Goode et al., 2016). Overall, Scl-binding sites could be mapped to 3061 genes (Table S2). Intersection of these genes with our mouse expression data from single knockout or DKO yolk sacs identified 114 genes that were differentially expressed in the DKO mice only (Fig. 6B). Of these, 33 genes were common to the 80 genes that were identified using the K562 ChIP-seq data (Table S2). Importantly, this subset included Gata1, Runx1, Zfpm1 and erythroid differentiation genes Gypc, Hmbs and Slc4a1.

Fig. 6.

Integration of ChIP-seq and RNA-seq data. (A) Venn diagram showing overlap of differentially expressed genes for ESclcKO, Lyl1KO and DKO cells with mouse homologs of the common SCL- and LYL1-binding sites identified in K562 cells. (B) Venn diagram showing overlap of differentially expressed genes with the genes nearest to Scl-binding sites in mouse hematopoietic progenitors. (C) De novo motif discovery of the 80 K562 ChIP-seq binding sites has identified significant enrichment for the GATA1 motif. (D) Targeted motif scanning has identified GATA, LYL1/SCL and RUNX1 motifs. (E) Intersection of LYL1, SCL, GATA1 and RUNX1 K562 ChIP-seq binding sites. (F) Venn diagram showing overlap of the 114 Scl-binding sites that have been identified in mouse hematopoietic progenitors with Gata1-, Lmo2- and Runx1-binding sites.

Fig. 6.

Integration of ChIP-seq and RNA-seq data. (A) Venn diagram showing overlap of differentially expressed genes for ESclcKO, Lyl1KO and DKO cells with mouse homologs of the common SCL- and LYL1-binding sites identified in K562 cells. (B) Venn diagram showing overlap of differentially expressed genes with the genes nearest to Scl-binding sites in mouse hematopoietic progenitors. (C) De novo motif discovery of the 80 K562 ChIP-seq binding sites has identified significant enrichment for the GATA1 motif. (D) Targeted motif scanning has identified GATA, LYL1/SCL and RUNX1 motifs. (E) Intersection of LYL1, SCL, GATA1 and RUNX1 K562 ChIP-seq binding sites. (F) Venn diagram showing overlap of the 114 Scl-binding sites that have been identified in mouse hematopoietic progenitors with Gata1-, Lmo2- and Runx1-binding sites.

Finally, de novo motif discovery of the shared SCL- and LYL1-bound sequences identified significant enrichment for GATA1 motifs (Fig. 6C), supporting our hypothesis that either SCL or LYL1 functions in concert with GATA1 to regulate erythroid growth and differentiation. This was further confirmed by targeted motif scanning (Fig. 6D), which verified enrichment for GATA1 (42% of bound sequences), and also identified the relative overrepresentation of LYL1/SCL (32% of bound sequences) and RUNX1 (26% of bound sequences) motifs. Of note, the majority of these sites were also bound by GATA1 with or without RUNX1 (Fig. 6E). Similarly, the 114 differentially expressed Scl targets identified in mouse hematopoietic progenitors (Fig. 6B) were also frequently bound by Lmo2 (97/114), Gata1 (58/114) and Runx1 (51/114) (Fig. 6F). Overall, these results suggest that shared Scl- and Lyl1-binding targets are also targets of Gata1, Lmo2 and Runx1.

DISCUSSION

Functional genetic redundancy is an evolutionary process that limits the impact of mutations on key developmental programs and enables a finer degree of control (Kafri et al., 2009). Robust primitive erythropoiesis is essential for the viability of the rapidly developing embryo and, in this context, we show that either Scl or Lyl1, two closely related bHLH TFs, are capable of maintaining primitive erythropoiesis. However, Lyl1KO embryos with a markedly reduced expression of Scl die at E10.5 because of the loss of primitive erythropoiesis. An integrated approach using expression analyses and ChIP shows that this functional redundancy is most likely explained by sharing common partner proteins and target genes including the master erythroid regulator Gata1.

We have previously shown that deletion of Scl in adult HSCs leads to a transient loss of erythroid progenitors that recovers within a few weeks (Hall et al., 2005). We postulated that Lyl1 was the TF that maintained erythropoiesis in the absence of Scl. To examine the functional role of Scl in primitive erythropoiesis, we used an erythroid-specific Cre transgenic mouse strain that can delete lox-P-flanked alleles in primitive erythroid cells from E8.5 (Drogat et al., 2010). The RNA-seq analyses performed at E9.5 demonstrated 10% residual Scl expression that was sufficient to allow embryonic development to adulthood. However, in the absence of Lyl1, this markedly reduced expression of Scl was unable to maintain primitive erythropoiesis, leading to the death of all embryos by E10-E11, a time in development that relies upon primitive erythropoiesis (Palis, 2014). There is, therefore, functional redundancy of Scl and Lyl1 for murine primitive erythropoiesis. Given that the Cre transgene is not active until E8.5, our experiments do not address the functional redundancy of Scl and Lyl1 for initiation of primitive erythropoiesis.

In light of our results, previous conclusions that Scl is essential for primitive erythropoiesis should be reconsidered. Deletion of Scl with the Tie2Cre transgene led to embryonic death with systemic edema and hemorrhage at around E13.5 (Schlaeger et al., 2005). Primitive erythroid cells showed a delay in maturation, leading to the conclusion that defects in erythropoiesis contributed to embryonic death. However, our results suggest that the loss of Scl in other lineages in which Tie2Cre is active, such as megakaryocytes and endothelium, might explain the embryonic death of Tie2Cre;SclΔ/Δ mice. A second study examined the phenotype of Scl knock-in mice (SclRER), which carry a germline mutation that prevents DNA binding (Kassouf et al., 2008). These mice had defects of both primitive and definitive erythropoiesis, with the majority (75%) of embryos dying between E14.5 and E17.5. This phenotype led to the conclusion that Scl DNA-binding was crucial for erythropoiesis. However, an alternate explanation for the embryonic lethality of the SclRER mice is a dominant-negative effect of the Scl DNA-binding mutant, which retains the ability to form a multimeric complex and therefore compete with Lyl1. A competitive model between the Scl DNA-binding mutant and Lyl1 could explain the later death of SclRER embryos and the partial penetrance in which 25% survived to adulthood with a mild anemia similar to ESclcKO mice. The ability of Lyl1 to bind a small subset (10%) of Scl target genes may also explain the ChIP-seq analyses of SclRER fetal livers, in which 20% of Scl-binding targets were retained in the absence of Scl DNA binding (Kassouf et al., 2010). In support of this hypothesis, we found significant overlap of common Scl/Lyl1-binding sites with the sites that can be bound by the Scl DNA-binding mutant that were identified in SclRER fetal livers (Table S3). Taken together, our results suggest that Scl is not essential for the maintenance of primitive erythropoiesis, as was previously thought.

The early death of DKO mice indicates that Lyl1 is the TF that is responsible for survival of the ESclcKO mice. Analysis of single-cell data at E7.75 revealed that 170/187 (91%) of erythroid cells express Scl and/or Lyl1 (Fig. 1A). This provides pools of cells that could explain the ongoing erythropoiesis in the absence of either factor at E10.5 into adulthood. The embryonic lethality of Lyl1KO mice that show markedly reduced Scl expression suggests that the minor cell population that lacks Scl and Lyl1 at E7.75 is either transient, insufficient to maintain survival at E10.5 or is an experimental artefact of single-cell technologies. The likely mechanism for in utero death of DKO embryos is transcriptional loss of Gata1, as Gata1 null embryos die at a similar developmental stage (Fujiwara et al., 1996). Reduced expression of Gata1 in DKO embryos cannot be explained by reduced numbers of erythroid cells because gene expression profiling was performed before significant loss of primitive erythropoiesis. Studies of embryonic stem cells shows an important role for Gata1 in erythroid survival and maturation (Weiss and Orkin, 1995). Our analyses of E9.5 DKO cells also suggests a role for Scl/Lyl1 in cell proliferation.

We identified 80 other direct target genes shared by Scl and Lyl1 that may also contribute to the embryonic lethality. However, the loss of many of these erythroid genes, including Klf1 and Zfpm1, is likely due to loss of the Scl-Gata1 and Lyl1-Gata1 complexes, given that the majority of common Scl/Lyl1-binding sites also bind Gata1 (Fig. 6C,D). The common binding sites for SCL, LYL1 and GATA1 is consistent with the presence of a multimeric transcriptional complex, although detailed analyses of the LYL1 complex in erythroid cells has not yet been reported. Given that Gata1 functions within a multiprotein complex that contains Scl, overexpression of Gata1 alone would not be sufficient to rescue the defects in DKO cells.

In summary, we have demonstrated that Lyl1 can compensate for the loss of Scl expression during primitive erythropoiesis, with functional redundancy due to shared DNA-binding targets including the crucial erythroid TF gene Gata1.

MATERIALS AND METHODS

Mice

Mice with homozygous lox-P-targeted Scl allele (SclcKO) (Hall et al., 2003) were crossed with erythropoietin receptor Cre-recombinase (EpoRCre) mice (Drogat et al., 2010) to generate EpoRCreSclcKO mice with lineage-specific deletion of Scl. These mice were then crossed with homozygous Lyl1 knockout mice (Lyl1KO) (Capron et al., 2006) to generate EpoRCreSclcKOLyl1KO mice, with both genes deleted in RBCs. The Animal Ethics Committee of the Alfred Medical Research and Education Precinct approved all animal experiments.

Blood counts

Mice were bled by puncturing the submandibular vein. Blood was then placed into Sarstedt Microvette collection tubes containing EDTA and full blood counts were performed on a Drew Scientific Hemavet system.

Embryo dissection and yolk sac collection

Embryos were collected at E9.5 to E15.5 and placed in PBS at room temperature and dissected with fine forceps under a dissecting microscope (Nikon SMZ1500). Photographs of embryos were taken with a Zeiss AxioCam MRC5 camera. Yolk sac cells were obtained by digestion at 37°C for 30 min in PBS with 7% fetal calf serum (FCS) and 1% penicillin/streptomycin collagenase/dispase (Sigma-Aldrich). Ice-cold PBS with 7% FCS and 1 mM EDTA solution was added to stop the digestion and the cells were then spun at 1500 rpm (300 g) for 5 min at 4°C. The yolk sac pellet was re-suspended in ice-cold PBS with 7% FCS and 1 mM EDTA, homogenized with vigorous pipetting and then filtered through a 40 μm nylon strainer. Samples were then used for cytospin, benzidine staining, RNA extraction and flow cytometry.

Flow cytometry

Flow cytometry and cell sorting was performed as previously described (Tremblay et al., 2010). Briefly 2×106 bone marrow cells were stained using BD Pharmingen antibodies against mouse CD71 (C2 ; 553266) and Ter119 (Ter-119; 561033) and BioLegend antibodies against mouse CD105 (Mj7/18; 120412) and CD150 (TC15-12F12.2; 115904). All antibodies were used at 1:500 except CD150 which was used at 1:100. Dead cells were excluded using propidium iodide staining. Cell sorting was performed using FACSAria (BD Biosciences) and FACS analysis was performed using CantoII, LSRII and LSR-Fortessa (BD Biosciences). Results were analyzed using DIVA and FlowJo software.

RNA extraction and qPCR

RNA was extracted using Trizol and the RNAeasy mini kit (Qiagen). RNA (1 μg) was used to make cDNA with the Transcriptor First Strand cDNA kit (Roche). qPCR was performed on a Lightcycler 480 II machine (Roche). Results were standardized to expression of the Hprt housekeeping gene.

RNA-seq

Total RNA from E9.5 yolk sacs was extracted for mRNA sequencing using Illumina HiSeq2500. Library preparation and sequencing was performed by Micromon (Monash University). Data was analyzed using an in-house developed pipeline, RNASik (v1.4.7) (monashbioinformaticsplatform.github.io/RNAsik-pipe). Raw reads were aligned using STAR (v2.5.2b) (Dobin et al., 2013) with GRCm38 reference genome from ENSEMBL. Gene counts were generated using feature counts in subread (v1.5.2) (Liao et al., 2014) with GRCm38.87 gene annotation from ENSEMBL. Differential expression analysis was performed in Degust (degust.erc.monash.edu) using LIMMA/edgeR/Voom. Public single-cell RNA-seq data: raw read count single-cell data were extracted from a recent study (Scialdone et al., 2016; gastrulation.stemcells.cam.ac.uk/scialdone2016) and analyzed using LIMMA(v3.32.2)/edgeR (v3.18.1)/Voom libraries in R Bioconductor v3.5 (bioconductor.org). Raw read counts were Voom-transformed into counts per million.

ChIP-seq analysis

The raw ChIP-seq reads were filtered for adapter contamination and low quality scores, and we also excluded reads in which more than 10% of bases were unknown. The following K562 ChIP-seq datasets were publicly available from GEO (SCL, GSE31477; RUNX1, GSE24777; GATA1, GSE70482). The ChIP-seq data for mouse hematopoietic progenitors was obtained from GSE69101 (Goode et al., 2016). Data processing was performed as previously described (Beck et al., 2013; Chacon et al., 2014; Wilson et al., 2010). In brief, reads were aligned to the human genome (hg19) using the software package BWA with standard parameters, resulting in 13,394,203 mapped reads for LYL1, 29,426,999 mapped reads for SCL, 8,033,900 mapped reads for RUNX1, 22,622,534 mapped reads for GATA1. Three publicly available peak finding programs, MACS (Feng et al., 2012), findPeaks (HOMER) (Heinz et al., 2010) and SPP (Kharchenko et al., 2008), were used to call peaks. Peaks called by at least two algorithms were identified as high-confidence binding sites for downstream analysis. De novo motif discovery was performed using MEME (Bailey and Elkan, 1994). High-confidence binding regions were assigned as regulatory regions to at most two genes using annotations provided by the GREAT analysis package (McLean et al., 2010). Homology information for human and mouse was downloaded from MGI (Blake et al., 2017) and we found that 16,824 human genes were associated with 17,196 mouse homologs. 1575 LYL1 and SCL target genes in K562 cells were mapped 1403 mouse homologs. RUNX1, GATA1 and TAL1 motifs were downloaded from the JASPAR database and occurrence searched using the FIMO software from the MEME suite. A bootstrapping approach was used to address the significance of combinatorial binding events between the TFs for all possible binding patterns. A lower estimate of 80,000 binding sites available for TF binding (Tijssen et al., 2011; Wilson et al., 2010) was used to estimate the background distribution of combinatorial binding events. The standardized z-score metric was then used to express the deviation of the combinatorial binding events from the expected mean (normalized by the standard deviation) of the background distribution (i.e. a positive z-score indicates an overlap more likely than random chance; a higher z-score indicates a more significant overlap).

Acknowledgements

The authors acknowledge Jacqueline E. Boyle for genotyping mice; staff at Monash ARL for animal husbandry; Jelena Kezic of Monash Histology Platform for processing and Haemotoxylin and Eosin staining of embryos and yolk sacs; and Geza Paukovics, Phil Donaldson and Eva Orlowski from AMREP flow cytometry facility for their assistance in flow cytometry. The authors would also like to thank Bertie Gottgens, University of Cambridge, for reading the manuscript and providing insightful feedback.

Footnotes

Author contributions

Conceptualization: D.B., J.E.P., C.S.T., D.J.C.; Methodology: C.S.T., D.J.C.; Formal analysis: S.K.C., J.S., Y.H., S.E.S., N.C.W., D.R.P., D.B., J.E.P., C.S.T., D.J.C.; Data curation: S.K.C., J.S., Y.H., S.E.S.; Writing - original draft: S.K.C., D.B., J.E.P.; Writing - review & editing: S.K.C., C.S.T., D.J.C.; Supervision: C.S.T., D.J.C.

Funding

This work was supported by a project grant APP1052313 (D.J.C. and J.E.P.) from the Australian National Health and Medical Research Council (NHMRC), a Leukaemia Foundation scholarship (S.K.C.), a Senior Medical Research Fellowship from the Sylvia and Charles Viertel Charitable Foundation (D.J.C.) and a Cancer Institute NSW Fellowship (D.B.). We also acknowledge funding from the Anthony Rothe Memorial Trust (J.E.P.), Cancer Australia (D.B), Gilead Sciences (D.B.) and a National Health and Medical Research Council Peter Doherty fellowship (D.B.).

Data availability

Data have been deposited in GEO under accession number GSE103789.

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Competing interests

The authors declare no competing or financial interests.

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