Biology subjects

Ratri, A.

Publications and source records attributed to Ratri, A..

4 recordsLinked to original sources

bioRxiv2020

DOT1L methyltransferase regulates the calcium influx in erythroid progenitor cells in response to erythropoietin

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of hematopoietic progenitor cells. An important downstream response of EPO signaling is calcium influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in c-KIT positive erythroid progenitor cells is regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development, including early erythropoiesis. We previously reported that Dot1L knockout (Dot1L-KO) hematopoietic progenitors in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we have detected a marked downregulation of Trpc6 gene expression in Dot1L-KO progenitor cells in the yolk sac compared to wildtype. However, the expression of Trpc2, the positive regulator of Ca2+influx, remained unchanged. The promoter and the proximal region of the Trpc6 gene loci exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. As the loss of DOT1L affects the expression of TRPC6, which inhibits Ca2+influx by TRPC2, Dot1L-KO progenitor cells in the yolk sac exhibit accelerated and sustained high levels of Ca2+influx. Such heightened Ca2+ levels might have detrimental effects on the development of hematopoietic progenitor cells in response to erythropoietin.

developmental biology↗

bioRxiv2020

The Role of DOT1L Methyltransferase Activity in Fetal Hematopoiesis

Early mammalian erythropoiesis requires the DOT1L methyltransferase. We demonstrated that loss of DOT1L in mutant mice resulted in lethal anemia during midgestation. The molecular mechanisms by which DOT1L regulates embryonic erythropoiesis have not yet been elucidated. In this study, a methyltransferase mutant mouse line (Dot1L-MM) was generated to determine whether the methyltransferase activity of DOT1L is essential for erythropoiesis. Dot1L-MM mice displayed embryonic lethality between embryonic days 10.5 and 13.5, similar to Dot1lL knockout (Dot1L-KO) mice. However, when examined at E10.5, unlike the Dot1L-KO, Dot1L-MM embryos did not exhibit evidence of anemia. In ex vivo hematopoietic differentiation cultures, Dot1L-KO and Dot1L-MM yolk sac (YS) cells both formed reduced numbers of myeloid, and mixed hematopoietic colonies. Erythroid colonies were able to be formed in numbers equal to wildtype embryos. Extensively self-renewing erythroblast (ESRE) cultures were established using YS cells from E10.5 embryos. Dot1L-KO and Dot1L-MM cells expanded significantly less than wild-type cells and exhibited increased cell death. Strikingly, Dot1L-KO and Dot1L-MM cells of YS origin exhibited profound genomic instability, implicating DOT1L methyltransferase activity in maintenance of the genome as well as viability of hematopoietic progenitors. Our results indicate that the methyltransferase activity of DOT1L plays an important role early murine hematopoiesis.

developmental biology↗

bioRxiv2020

SATB homeobox 1 regulated genes in the mouse ectoplacental cone are important for placental development

SATB homeobox 1 (SATB1) is abundantly expressed in the stem-state of trophoblast cells but downregulated during trophoblast differentiation. It is also expressed in high levels in the mouse ectoplacental cones (EPCs). We detected that SATB1 is involved in maintaining the self-renewal of trophoblast stem cells and inhibiting trophoblast differentiation. In this study, we have identified SATB1-regulated genes in the mouse EPC and analyzed their potential functions. A total of 1618 differentially expressed genes were identified in Satb1null EPCs by mRNA sequencing. Remarkably 90% of the differentially expressed genes were found to be upregulated in Satb1null EPCs suggesting a transcriptional repressor role of SATB1 in mouse trophoblast cells. Ingenuity Pathway Analyses demonstrated that the differentially expressed genes in Satb1null EPCs are particularly linked to WNT and TGF{beta} signaling pathways, which regulate self-renewal of stem cells and cell differentiation. Moreover, twenty-six of the EPC genes that are known to be involved in placental development including Eomes, Epas1, Fgfr2, Cdkn1c, and Plac9 were found dysregulated in Satb1null EPCs due to the loss of SATB1 expression. These genes are particularly involved in the formation of labyrinthine zone. Our results emphasize that SATB1-regulated genes in the mouse EPC contribute to key roles in the regulation of trophoblast differentiation and placental development.

developmental biology↗

bioRxiv2020

A long-range chromatin interaction regulates SATB homeobox 1 gene expression in trophoblast stem cells

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages determined during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated active state of the promoter. A novel cis-regulatory region with active histone marks was identified [~]21kbp upstream of variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromatin conformation capture confirmed looping of this regulatory region into the promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close vicinity of SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

developmental biology↗