Gata1 expression driven by the alternative HS2 enhancer in the spleen rescues the hematopoietic failure induced by the hypomorphic Gata1low mutation

Abstract

Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1low mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis

“…23 Splenectomy, however, prevents development of MF in heterozygous females, suggesting that spleen provides a supportive microenvironment for Gata1 low stem cells. 30 This concept was recently validated by the observation that the spleen of PMF patients is uniquely enriched for PMF stem cells. 50 The great numbers of PMF stem cells observed in the spleen of the patients and the different TGF-b1 alteration signatures of BM and spleen described here suggest a model for the development of MF, according to which TGF-b1 released by the MK progeny of MF stem cells induce different TGF-b1 expression signatures in BM and spleen, which suppress normal hematopoiesis in BM and activate MF hematopoiesis in spleen (supplemental Figure 4).…”

“…Cycle thresholds (Ct) were calculated with the SDS software and messenger RNA (mRNA) levels expressed in relative units using 2 2DCt (D Ct 5 target gene Ct 2 b-2 microglobulin Ct) values observed in the untreated Gata1 low group as reference. 30 TGF-b pathway profiles were obtained with the Mouse TGF-b RT 2 Profiler PCR array kit (SABiosciences, Frederick, MD) as described by the manufacturer. Differences in gene expression >2-fold between untreated or vehicletreated or SB431542-treated Gata1 low mice and wild-type controls were identified using the PCR Array Data Analyses Web portal (SABiosciences) and considered statistically significant with a P , .05 by a 2-tailed, Student t test.…”

“…30 These alterations were validated by performing similar analysis on Gata1 low mice treated with the TGF-b1 inhibitor SB431542 or vehicle, as control. SB431542 treatment significantly reduced by ;20% TGF-b1 mRNA levels ( Figure 2) and by 2-to 4-fold the intensity of TGF-b1 staining of MK both in BM and spleen (Figure 1A-C; Table 2).…”

“…However, due to increased BM cellularity (Figure 4B), the total number of progenitor cells in BM increased by 5-fold after SB431542 treatment ( Figure 5B). Progenitor cells from BM of Gata1 low mice express an altered profile characterized by reduced expression of Gata1 and the cell cycle regulator p27 kip ( Figure 5C) 30,39 . SB431542 treatment, but not vehicle treatment, normalized the levels of both Gata1 and p27 kip1 mRNA expressed by these cells.…”

The Human GATA1 Gene Retains a 5′ Insulator That Maintains Chromosomal Architecture and GATA1 Expression Levels in Splenic Erythroblasts

“…In this regard, it is interesting to note that the mouse spleen has a distinct hematopoietic microenvironment that plays a crucial role in stress erythropoiesis (42). A recent report showed that mGata1 expression is more severely repressed in splenic CD71 high erythroblasts than in the corresponding population in the bone marrow of Gata1 low mutant mice in which G1HE/HS1, a potent cis-acting enhancer element in the Gata1 gene, is deleted from the endogenous locus (43). Showing very good agreement with these observations, the findings of our present study show that hGATA1 gene expression is significantly diminished in splenic erythroblasts upon the loss of the chromosome architecture in CTCFmut-hG1B transgenic mice under conditions of hemolytic anemia.…”