Non-coding RNA LEVER sequestration of PRC2 can mediate long range gene regulation.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA, LEVER, which mapped 236 kb upstream of the ß-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and ß-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.

Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal ß-like globin genes.

The clinical symptoms of hemoglobin disorders such as ß-thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ-globin after birth. This knowledge has driven the discovery of important regulators that silence γ-globin postnatally. Improved understanding of the γ- to ß-globin switching mechanism holds the key to devising targeted therapies for ß-hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183-kb human ß-globin locus, in which the (G)γ- and ß-globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)-mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ-globin, measured by DsRed fluorescence and qRT-PCR (P<0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε-globin, (up to 20% of total ß-like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ-globin expression (up to 90% of total ß-like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human ß-like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal ß-like globin genes in the adult erythroid environment.

Analysis of primitive erythroid cell proliferation and enucleation using a cyan fluorescent reporter in transgenic mice.

Primitive erythropoiesis is a vital process for mammalian embryonic development. Here we report the generation and characterization of a new transgenic mouse line that expresses a histone H2B-CFP fusion protein in the nuclei of primitive erythroid cells. We demonstrate the potential of this ε-globin-histone H2B-CFP line for multicolor imaging and flow cytometry analysis. The ε-globin-H2B-CFP line was used to analyze the cell cycle distribution and proliferation of CFP-expressing primitive erythroblasts from E8.5-E13.5. We also evaluated phagocytosis of extruded CFP-positive nuclei by macrophages in fetal liver and placenta. The ε-globin-H2B-CFP transgenic mouse line adds to the available tools for studying the development of the primitive erythroid lineage.

Decitabine increases fetal hemoglobin in Papio anubis by increasing γ-globin gene transcription.

OBJECTIVE: The mechanism responsible for increased fetal hemoglobin levels following decitabine treatment remains controversial. These experiments were performed to evaluate the role of transcriptional vs. translational mechanisms in the ability of decitabine to increase fetal hemoglobin levels in vivo. MATERIALS AND METHODS: Three normal, nonanemic baboons were treated with decitabine subcutaneously (0.5 mg/kg/d) for 10 days. The effect of decitabine on globin chain synthesis and globin messenger RNA levels was measured in pre- and posttreatment bone marrow aspirates by biosynthetic radiolabeling with [(3)H] leucine followed by separation of globin chains by high-performance liquid chromatography, and real-time polymerase chain reaction, respectively. The effect on DNA methylation of the ɛ- and γ-globin gene promoters was determined by bisulfite sequence analysis. RESULTS: Decitabine treatment of normal, nonanemic baboons induced similar increases in the γ/γ+ß chain synthetic ratio and the γ/total ß-like globin RNA ratio and also increased expression of ɛ-globin transcripts. Increased expression of ɛ- and γ-globin was associated with decreased DNA methylation of the ɛ- and γ-globin gene promoters. CONCLUSIONS: Decitabine increases fetal hemoglobin in vivo by transcriptional activation of the γ-globin gene.

Embryo-fetal erythroid megaloblasts in the human coelomic cavity.

The coelomic cavity is part of the extraembryonic mesoderm, surrounding amniotic cavity, embryo, and yolk sac in the early gestation. It is now believed to represent an important transfer interface and a reservoir of nutrients for the embryo. Coelocentesis by ultrasound-guided transvaginal puncture offers an easier access to the early human embryo, from 28 days post-fertilization. However, despite some studies about its biochemical composition being reported, our knowledge about the presence of cellular elements and their quality in this compartment are still limited. Here we studied human coelomic fluids sampled from 6.6 (48 days) to 10 weeks of gestation, demonstrating the presence of functional embryonic erythroid precursors, that is, megaloblasts in the coelomic cavity. The ease of access of the coelomic cavity could allow the development of novel strategies for diagnostic or therapeutic purposes by ultrasound imaging and ultrasound-guided puncture.

DNase I hypersensitivity and epsilon-globin transcriptional enhancement are separable in locus control region (LCR) HS1 mutant human beta-globin YAC transgenic mice.

Expression of the five beta-like globin genes (epsilon, Ggamma, Agamma, delta, beta) in the human beta-globin locus depends on enhancement by the locus control region, which consists of five DNase I hypersensitive sites (5'HS1 through 5'HS5). We report here a novel enhancer activity in 5'HS1 that appears to be potent in transfected K562 cells. Deletion analyses identified a core activating element that bound to GATA-1, and a two-nucleotide mutation that disrupted GATA-1 binding in vitro abrogated 5'HS1 enhancer activity in transfection experiments. To determine the in vivo role of this GATA site, we generated multiple lines of human beta-globin YAC transgenic mice bearing the same two-nucleotide mutation. In the mutant mice, epsilon-, but not gamma-globin, gene expression in primitive erythroid cells was severely attenuated, while adult beta-globin gene expression in definitive erythroid cells was unaffected. Interestingly, DNaseI hypersensitivity near the 5'HS1 mutant sequence was eliminated in definitive erythroid cells, whereas it was only mildly affected in primitive erythroid cells. We therefore conclude that, although the GATA site in 5'HS1 is critical for efficient epsilon-globin gene expression, hypersensitive site formation per se is independent of 5'HS1 function, if any, in definitive erythroid cells.