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Our body responds to environmental stress by changing the expression levels of a series of cytoprotective enzymes/proteins through multilayered regulatory mechanisms, including the KEAP1-NRF2 system. While NRF2 upregulates the expression of many cytoprotective genes, there are fundamental limitations in short-read RNA sequencing (RNA-Seq), resulting in confusion regarding interpreting the effectiveness of cytoprotective gene induction at the transcript level. To precisely delineate isoform usage in the stress response, we conducted independent full-length transcriptome profiling (isoform sequencing; Iso-Seq) analyses of lymphoblastoid cells from three volunteers under normal and electrophilic stress-induced conditions. We first determined the first exon usage in KEAP1 and NFE2L2 (encoding NRF2) and found the presence of transcript diversity. We then examined changes in isoform usage of NRF2 target genes under stress conditions and identified a few isoforms dominantly expressed in the majority of NRF2 target genes. The expression levels of isoforms determined by Iso-Seq analyses showed striking differences from those determined by short-read RNA-Seq; the latter could be misleading concerning the abundance of transcripts. These results support that transcript usage is tightly regulated to produce functional proteins under electrophilic stress. Our present study strongly argues that there are important benefits that can be achieved by long-read transcriptome sequencing.

The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3′ region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal Epo regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

Erythropoietin (Epo) is secreted from the liver and kidney, where Epo production is strictly regulated at the transcriptional level in a hypoxia- and/or anemia-inducible manner. Here, we examined the in vivo function of the enhancer located 3′ to the Epo gene (EpoE-3′). Reporter transgenic-mouse analyses revealed that the EpoE-3′ enhancer is necessary and sufficient for the liver-specific and hypoxia-responsive expression of the gene after embryonic day 14.5 (E14.5). However, the enhancer is dispensable for Epo gene expression in the kidney and early-stage embryonic liver. Genetic removal of EpoE-3′ from the endogenous Epo gene resulted in mice with severe anemia at late embryonic and neonatal stages due to defects in hepatic erythropoiesis, but early hepatic and splenic erythropoiesis was not affected. The mutant mice recover from the anemia in the juvenile period when major Epo production switches from the liver to the kidney. These results demonstrate that EpoE-3′ is necessary for late hepatic erythropoiesis by specifically supporting paracrine production of Epo in the liver. In contrast, Epo production in the kidney utilizes distinct regulatory machinery and supports erythropoiesis in the bone marrow and spleen in adult animals.

The developmental origin of definitive hematopoietic precursors within a conceptus has been the subject of considerable study and debate. Several lines of evidence suggest that definitive hematopoietic progenitors originate from the para-aortic splanchnopleura (P-Sp) within the embryo proper (9, 26, 33). Lending credence to this hypothesis, Cumano et al. (5) reported that the caudal intraembryonic splanchnopleura of E7.5 to 8.5 murine embryos could give rise to mixed lymphoid-myeloid colonies in vitro. Similarly, human P-Sp cells produced mixed lymphohematopoietic colonies after culture on murine stromal cells or in fetal thymic organ culture. In contrast, human yolk sac (YS) cells did not produce lymphocytes in culture (55). Furthermore, P-Sp-derived but not YS-derived cells from ≈E8.5 embryos generated hematopoietic cells capable of long-term reconstitution in sublethally irradiated Rag2γc−/− adult mice (6). The developmental importance of the embryonic mesoderm in hematopoietic ontogeny has also been demonstrated in the chicken and frog (8, 59, 60). It has been alternatively postulated that definitive hematopoietic stem cells migrate en route from the yolk sac (15, 42, 64) to the fetal liver and eventually populate the adult hematopoietic compartment. However, hematopoietic precursors isolated from E7.5 to 8.5 yolk sacs were reportedly incapable of producing lymphoid-myeloid colonies in vitro (5). While one group had reported successful hematopoietic repopulation using E8.5 YS cells in sublethally irradiated mice (25), these cells were generally defective in reconstituting the hematopoietic panoply of adult mice exposed to sublethal irradiation (6, 27, 33). The vertebrate GATA transcription factor family comprises six members (reviewed in references 20, 39, 40, and 63) and each is expressed in a tissue- and developmental stage-specific manner. Of the six GATA factors, GATA-1, GATA-2, and GATA-3, contribute to different aspects of hematopoietic development and are categorized as the “hematopoietic” GATA factors (39), although GATA-2 and GATA-3 additionally contribute to numerous aspects of organ and tissue development outside of hematopoiesis. GATA-1 is necessary for the maturation of primitive and definitive erythroid cells (49, 51), megakaryocytes (61), eosinophils (13), and mast cells (12), whereas GATA-3 plays an indispensable role in the development of T cells (56, 65). In contrast, GATA-2 is indispensable for all hematopoiesis, since gene targeted loss of Gata2 in the mouse germ line leads to embryonic lethality as a consequence of the failure to expand the progenitor pool (58). Consistent with this conclusion, analysis of chimeric embryos generated using marked Gata2−/− embryonic stem (ES) cells indicated that these cells failed to contribute to any hematopoietic lineage. Several lines of evidence suggest that GATA-2 is expressed in pluripotent hematopoietic stem cells (41) and possibly in hemangioblasts, believed to represent the earliest common precursor of the hematopoietic and endothelial lineages (2, 3, 6, 40). Gata2 haploinsufficiency reduced the number of early hematopoietic stem cells and impaired the quality of both embryonic and adult hematopoietic stem cells, demonstrating that GATA-2 achieves its normal function only with a diploid contribution of factor (22). Interestingly, GATA-2 appears to be dispensable for the development of endothelial cells, as no vascular defects are evident in Gata2-deficient embryos (58). However, the precise contribution of GATA-2 to the earliest events that define hematopoiesis remains unclear. We previously showed that the mouse Gata2 gene has two first exons (28). The gene-distal first exon (IS) is specifically expressed in hematopoietic and neural cells, whereas the gene-proximal first exon (IG) is transcribed in almost all Gata2-expressing cells (29). Differential utilization of two distinct first exons has also been demonstrated for the human and chicken Gata2 genes (37, 44). We also reported that the murine Gata2 gene contains a number of hematopoietic regulatory elements scattered over more than 250 kbp of the locus, since a 250-kbp yeast artificial chromosome transgene was capable of rescuing the hematopoietic lethal deficiency in Gata2 null mutant embryos (66). Detailed investigation of Gata2 gene-proximal sequences indicated that a 7-kbp fragment flanking the IS exon could recapitulate Gata2 expression in early embryonic hematopoietic tissues (29). Taken together, these studies implicated the presence of critical cis-acting regulatory elements in the immediate vicinity of the IS promoter required for Gata2 expression in developmentally naive hematopoietic cells, while much more distal regulatory information may be required for complete hematopoietic progenitor differentiation. In this study, we investigated four aspects of GATA-2 regulation in early hematopoietic cells. First, we identified a 3.1-kbp domain 5′ to the IS exon that recapitulates Gata2 gene expression in the P-Sp and YS and named this region the Gata2 gene early hematopoietic regulatory domain (G2-EHRD). Second, we determined that G2-EHRD-directed green fluorescent protein (GFP) reporter transgenes accurately reflected the endogenous Gata2 expression pattern. When we performed detailed characterization of the GFP-marked cells in the P-Sp and yolk sac, we found that the P-Sp-derived and G2-EHRD-directed GFP+ cells could differentiate into hematopoietic as well as endothelial cells when cultured on OP9 stromal cells. Finally, we elucidated a set of indispensable GATA motifs within G2-EHRD, clarifying in vivo a key Gata2 cis-regulatory element that is active in early embryonic hematopoietic tissues. Thus, this study defines cis elements that contribute to the regulatory mechanisms underlying Gata2 expression in progenitor cells that appear to be able to adopt either a hematopoietic or endothelial developmental fate.