SUPPLEMENTARYINFORMATION

Supplemental Material and Methods

Primers for RT-qPCR are reported in table 1s.

Forward primer / Reverse primer
AHSP / GGATCAGCAGGTCTTTGATG / AGAGTACTCAGCTCTTGCTG
ALAS2 / GACTGGTGACCAGGCTTTCG / TGGTCCTGTTTCTTCTCCATGA
ALAD / ACGTGGGCTTGCGCTTTTAG / TCAGCTGCAGAGCCCTGTTC
ERMAP / GCCCGATGTCAGACCACCCTGCCTG / CAGGGACTTGGTCTCTCCATATGAAC
EPB4.9 / TGCTCAAGACCCAAGGCTTA / TCCTATCTGGTTTTGCCTGG
E2F2 / CCCCAAAACCCCCAAGTCT / ACTCGCTCAGGAGGTAAATGAACT
CDKN2C (p18) / CCACCTCCCTGTGGTGGAGT / GTGGCTCCCCCAACCCCATT
CDKN2B (p27) / AGTGTCCAGGGATGAGGA / GGGAACCGTCTGAAACAT
Bcl2l1 / AATGACCACCTAGAGCCTTGGA / TCGGCTGCTGCATTGTTC
Bcl2l11 / TGACTCTGATTCTCGGACTGAGAA / CAGAAGGTTGCTTGGCCATT
Mouse alpha / CACCACCCTGCCGATTTC / CTCACAGAGGCAAGGAATTTGTC
Hbb-b1 / TTTAACGATGGCCTGAATCACTT / CAGCACAATCACGATCATATTGC
HPRT / GCAGTACAGCCCCAAAATGG / AACAAAGTCTGGCCTGTATCCAA

Table1s

Flow cytometry Analysis

Freshly isolated fetal liver cells from E14.5 mice were collected and stained with anti-mouse CD45-PE, anti-mouse TER119-FITC (BD-Bioscience), anti-mouse IFNAR1-APC (Biolegend), anti-mouse CD71-PE, anti-mouse F4/80-FITC (BD-Bioscience) at a final concentration 1:100. Data were collected on a FACSCANTO flow cytometer (BD-Bioscience) and analyzed with FACSDiva software Version 6.1.3 (BD Biosciences) and FloJo V7.6.5.

SupplementalResults

Ifnar1 Flow cytometry Analysis

Klf1+/+, Klf1-/-from freshly isolated fetal liver cells were collected and analyzed by flow cytometry to verify Ifnar1 expression in erythroblasts during erythropoiesis and potential differences in Ifnar1 expression betweenWT and Klf1KO at E14.5. No significant difference in Ifnar1expression was observed either during erythropoiesis (not shown) or between Klf1+/+ andKlf1-/-fetal liver cells (as shown in Fig1S )

Fig.1s

CD44, CD71 and Ter119 Flow cytometry Analysis

Freshly isolated fetal liver cells from Klf1+/+/Ifnar1+/+, Klf1-/-/Ifnar1+/+ and Klf1-/-/Ifnar1-/- mice were collected and analyzed by flow cytometry to assess the level of expression of erythropoiesis key markers (CD44, CD71 and Ter119) and a possible rescue of the expression level in Klf1-/-/Ifnar1-/- compared to Klf1-/-/Ifnar1+/+ mice. As shown in Fig 2s, CD44 and CD71 markers do not show any significant difference between Klf1-/-/Ifnar1+/+ and Klf1-/-/Ifnar1-/- mice. Ter119 maker is not expressed in Klf1 null mice and no rescue is observed in Klf1/Ifnar1 double KO mice.

Fig. 2S

F4/80 Flow cytometry Analysis

Freshly isolated fetal liver cells from Klf1+/+/Ifnar1+/+, Klf1-/-/Ifnar1+/+ and Klf1-/-/Ifnar1-/- mice were collected and analyzed by flow cytometry to quantitate the relative number of F4/80 positive cells (three sample per genotype).Data have been normalized towards Klf1+/+/Ifnar1+/+ (WT) control. We observed a significant increase of F4/80 positive macrophages in Klf1-/-Ifnar1-/-compared to Klf1-/-Ifnar1+/+,(0.68 +/-SD 0.27vs0.27+/-SD 0.014 respectively, p=0,042). (Figure 3S).

Fig. 3S

Gene expression of Klf1 target genes is not significantly improved.

Klf1 regulates the development of erythroid cells trough the activation and regulation of several erythroid specific genes. Anemia and the consequential dead of Klf1 null mice is mainly explained by its role in the activation of genes for Hbb-b1 (-globin), Alpha Stabilizing Protein (AHSP), heme biosynthesis (e.g ALAS2), cell cycle regulator (e.g E2F2), component of erythroid cytoskeleton (e.g Epb4.9) and erythroid specific protein ( e.g ERMAP).

The down-regulation of these genes results in reduced hemoglobin production, exacerbate globin imbalance, membrane fragility and impaired proliferation. In order toevaluate if a recovery of the expression of the genes aforementioned would contribute to the amelioration of definitive erythropoiesis seen in Klf1-/-Ifnar1-/- mice we have carried out RT-qPCR assay on E 14.5 fetal liver cells. Our results (Fig. 4S) show that there is not significant differences in the relative expression of Hbb-b1 (p=0.24), AHSP (p=0.13), ALAS2 (p=0.46), and E2F2 (p=0.90) genes between Klf1-/- Ifnar1+/+andKlf1-/-Ifnar1-/-mice. Only Epb4.9 and ERMAP genes show a small improvement of their expression (from 6 to 8% and from 11 to 19%, p= 0.034 and p=0.011 respectively).

The failure of the orthochromatic erythroblast to enucleate is one of the landmark of the Klf1 null phenotype. Biekers JJ and coworkers (7) have recently shown that Klf1 null late erythroblast fail to enucleate because they continue to cycle due to the low expression of p18 (Cdkn2c) and p27 (Cdkn1b), direct Klf1 target genes. Here we show that enucleation and production of mature reticolocytes is partially restored in Klf1-/-Ifnar1-/- mice. We have evaluated the expression level of p18 and p27 genes by RT-qPCR on E14.5 fetal live embryos. As shown in Fig 3S there is not significant differences in the relative expression of these two genesbetween Klf1-/- Ifnar1+/+and Klf1-/-Ifnar1-/- mice(p=0,95 and p=0,21 respectively).

In summary these results suggest that the improvement of definitive erythropoiesis and enucleation in Klf1-/-Ifnar1-/- compared to Klf1-/-Ifnar1+/+ is not due to a significant increase in the expression of Klf1 direct target genes. The modest increase of Epb4.9 and ERMAP may be due to a (unknown) positive feedback to the recovery of erythroblast cell morphology associated to the improved erythropoiesis.

Fig.4S