MiRNA 451 is required for maintenance of erythroid cells in zebrafish. (A) WISH analysis for expression of erythroid markers in wild-type and miR 451 morphant embryos at ≈24 hpf. At this stage, committed erythroid cells are relatively immature and blood circulation is not yet established. The expression of erythroid markers in morphant embryos is unchanged, indicating normal specification of primitive erythropoiesis. The morphant embryos showed strongly reduced expression of miR 451 (SI Fig. 10). (B) WISH analysis for expression of βe1-globin in wild-type and miR 451 morphant embryos at ≈48 hpf. At this stage, mature erythroid cells have entered the circulation. The ducts of Cuvier on the yolk (square bracket, 1) and the cardinal vein (arrow, 2) are indicated, and higher-magnification views are shown in Lower. (C) o-dianisidine staining for hemoglobin in morphant embryos at 48 hpf.

Predicted miR 144/451 mRNA targets are overrepresented among repressed genes during later stages of GATA-1-induced erythroid maturation. Messenger RNAs that are repressed during GATA-1-induced erythroid maturation of G1E cells were identified from a previously described dataset (9). We focused on repression between 7 and 21 h after GATA-1 activation, when miR 144/451 becomes induced and reaches maximal levels (see Fig. 1A). The repressed transcripts were examined for expression of predicted miR 144 and 451 targets identified by miRBase (http://microrna.sanger.ac.uk/targets/v4). Fisher's exact test was used to evaluate whether predicted miRNA targets are overrepresented among repressed mRNAs relative to nonrepressed ones. One-sided P values indicating the significance of differences between potential targets and nontargets are shown. An odds ratio of 1.0 indicates that targets are more likely to be repressed within the tested time interval.

Enhancer activity of miR 144/451 preCRMs. (A) Design of the reporter construct. PreCRMs were positioned upstream of a γ-globin gene promoter linked to a firefly luciferase cDNA. The bent arrow indicates the start of the luciferase open reading frame. (B) Luciferase activities, reported as the fold increase over that of the parent plasmid, are shown from three separate transient transfection experiments, represented as separate bars. Error bars indicate standard deviation from quadruplicate luciferase determinations from a single experiment. Reporter assays were performed in human K562 erythroleukemia cells and in MEL cells treated with HMBA to induce erythroid maturation. Firefly luciferase activities are normalized to Renilla luciferase activities expressed from a cotransfected control plasmid. The DNA segment Zfpm1R13 is a positive control for a known enhancer (30).

MiRNAs 144 and 451 are abundant in erythroid tissues and GATA-1-regulated. (A) Northern blot showing induction of miR 144 (Top) and miR 451 (Middle) after activation of a GATA-1-estrogen receptor fusion protein in G1E-ER4 murine erythroblasts. Five micrograms of total cellular RNA was analyzed in each lane. (Bottom) Ethidium bromide staining of 5.8S and 5S rRNAs used as a loading control. (B) Multitissue Northern blot of miR 144 and miR 451 expression in mice. MiR 194, which is expressed in nonhematopoietic tissues, was examined as a control. Five micrograms of total cellular RNAs from the following murine tissues were analyzed: Bl, whole blood; Br, brain; Ht, heart; Kn, kidney; Lv, liver; Lg, lung; Mu, muscle; Sp, spleen; Te, testes. (C) Splenocytes were fractionated according to expression of the erythroid-specific surface antigen Ter119 and analyzed for miR 451 expression by Northern blotting. The third lane labeled “oligo” represents 0.1 pmol of a miR 451“sense” DNA oligonucleotide used as a hybridization control. (D) Expression of miR 451 and miR 144 in zebrafish embryos at 26 hpf. Wild-type and gata-1⁻ (vlad tepes) embryos were analyzed by WISH by using miR 144 and miR 451 probes. Expression of both miRNAs was observed in the blood island of the ICM, shown in the boxed regions. Higher-magnification views of the ICM (arrow) are displayed in the Insets. Expression of miR 451 and miR 144 is markedly reduced in the ICM of vlad tepes embryos.

The miR 144/451 locus is directly activated by GATA-1. (A) Features of the miR 144/451 locus and 5′ flanking DNA. A 10-kb region of mouse chromosome 11 (mm8 assembly) is annotated with the DNA encoding miRNAs (thin black rectangles), the transcription start (bent arrow), and the 3′ end of the adjacent Eral1 gene located telomeric to the miRNA locus. PreCRMs (27) and amplicons used in ChIP assays in B and C are shown as rectangles above and below the line, respectively, with the positions of the amplicons relative to the transcription start given in kilobases (SI Table 3). The −2.8-kb preCRM validated in ChIP (B–D) and enhancer assays (Fig. 3) is shown in black. The positions of GATA consensus binding motifs (WGATAR), either conserved in mammals or present only in rodents, are indicated as vertical lines. The track labeled “regulatory potential” plots sequence similarity to alignment patterns in known regulatory regions (28). (B) Quantitative ChIP analysis of the locus in G1E (no GATA-1) and G1E-ER4 cells treated with estradiol (E2) for 24 h (activated GATA-1). The relative occupancies of GATA-1, FOG-1, and GATA-2 are indicated as vertical bars. As a negative control, ChIP experiments were performed with isotype-matched preimmune IgG. The bar graphs show averages of three independent ChIP experiments. Error bars represent standard deviation. (C) Quantitative ChIP analysis for GATA-1 and FOG-1 in primary fetal liver cells (embryonic day 14) and MEL cells induced to mature with HMBA, performed as in B. (D) Quantitative ChIP analysis of RNA pol II binding to the miR 144/451 locus, performed as in B. Amplicons labeled 0.7 and 1.0, not shown in A, extend into the transcribed region and are designated according to the distance (in kilobases) from the start of transcription.