Activation of the VEGFR1 Chromatin Domain

et al. 2009

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During early mammalian development, genesis of the first two cell lineages, inner cell mass (ICM) and trophectoderm (TE), is dependent upon functions of key transcription factors that are expressed in a regulated and spatially restricted fashion. In this study, we demonstrate that during early mouse development, mRNA expression of transcription factor GATA3 is induced at the 4-cell stage and is consistently present during pre-implantation embryonic development. Interestingly, at the blastocyst stage, Gata3 mRNA is selectively up-regulated within the TE lineage, and GATA3 protein is abundantly present only in the TE but not in the ICM. Using mouse trophoblast stem cells (TS cells) as a model, we found that, knockdown of GATA3 by RNA interference (RNAi) down-regulates expression of caudal-type homeobox 2 (CDX2), a key regulator of the TE lineage. Chromatin immunoprecipitation (ChIP) analyses revealed that, in TS cells, GATA3 directly regulates Cdx2 transcription from a conserved GATA motif at the intron 1 region of the Cdx2 locus. ChIP analyses with mouse blastocysts also detected GATA3 occupancy at intron 1 of the Cdx2 locus. In addition, downregulation of GATA3 in pre-implantation mouse embryos reduces Cdx2 expression and inhibits morula to blastocyst transformation. Our results indicate a novel function of GATA3, in which it is selectively expressed in TE, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation. Genesis of the trophectoderm (TE)2 and inner cell mass (ICM) lineages during early mouse development appears to occur in two stages (1-3). First, cells are allocated to different inside and outside positions via asymmetric divisions. Then, the cells in these different positions become specified, and they become committed to restricted developmental fates. Outside cells become committed to the TE, and inside cells become ICM. Development of ICM and TE is regulated by key transcription factors that specify TE and ICM cell fate, and CDX2 has been implicated in this process (4 -6). Multiple studies indicated the importance of CDX2 in TS cell proliferation, proper function of TE, and successful implantation of blastocyst (5-8). However, molecular mechanisms that regulate Cdx2 expression in trophoblast cell lineages are poorly understood. Two other transcription factors, eomesodermin (Eomes) and TEA domain family member 4 (TEAD4), are also implicated in TE development. Mutation studies showed that the lack of Eomes also arrests blastocyst development (9). However, Cdx2 is still expressed in Eomes mutants (5). Tead4 mutants show more severe phenotypes than Cdx2 mutants and are characterized by loss of Cdx2 expression (10, 11). However, unlike Cdx2, Tead4 expression is not restricted to the TE lineage during pre-implantation development indicating that additional regulatory mechanisms are involved for the restricted expression of Cdx2 in TE lineage.Earlier, we found that, among the six members (GATA1-6) of GATA family of transcription factors, only GATA3 is abunda...

Section: Methodsmentioning

confidence: 99%

GATA3 Is Selectively Expressed in the Trophectoderm of Peri-implantation Embryo and Directly Regulates Cdx2 Gene Expression

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et al. 2009

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“…Cell Culture and Reagents-YSECs and HUVECs were maintained in medium 200 (M200; Invitrogen) as described earlier (15,16). For regular maintenance (used as control), the cells were cultured in M200, supplemented with an endothelial growth supplement (Invitrogen) containing FGF2 (3 ng/ ml), EGF (10 ng/ml), hydrocortisone (1 g/ml), and heparin (10 g/ml) along with FBS (final concentration, 2%).…”

Section: Methodsmentioning

confidence: 99%

“…Our earlier studies with mouse yolk sac endothelial cells (YSECs) and human umbilical vein endothelial cells (HUVECs) showed that Vegfr1 transcription is highly induced in endothelial cells in response to angiogenic signals mediated by a growth supplement containing FGF2 and EGF (15,16). We also showed that transcription factors ETS1 and hypoxia-inducible factor 2␣ function in a combinatorial fashion to directly mediate the transcriptional induction of Vegfr1 in endothelial cells (16). However, the role of chromatin-associated mechanisms like the importance of a specific histone modification in the transcriptional regulation of Vegfr1 or other key angiogenic genes has never been addressed.…”

mentioning

confidence: 99%

Regulation of Angiogenesis by Histone Chaperone HIRA-mediated Incorporation of Lysine 56-acetylated Histone H3.3 at Chromatin Domains of Endothelial Genes

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et al. 2010

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“…Lentiviral supernatants were produced in human embryonic kidney-293T cells by transfection with calcium phosphate as described earlier (33). Lentiviral supernatants were collected after 24 and 48 h of incubation.…”

Section: Methodsmentioning

confidence: 99%

Context-dependent Function of Regulatory Elements and a Switch in Chromatin Occupancy between GATA3 and GATA2 Regulate Gata2 Transcription during Trophoblast Differentiation

Rumi

et al. 2009

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GATA transcription factors are important regulators of tissue-specific gene expression during development. GATA2 and GATA3 have been implicated in the regulation of trophoblastspecific genes. However, the regulatory mechanisms of GATA2 expression in trophoblast cells are poorly understood. In this study, we demonstrate that Gata2 is transcriptionally induced during trophoblast giant cell-specific differentiation. Transcriptional induction is associated with displacement of GATA3-dependent nucleoprotein complexes by GATA2-dependent nucleoprotein complexes at two regulatory regions, the ؊3.9-and ؉9.5-kb regions, of the mouse Gata2 locus. Analyses with reporter genes showed that, in trophoblast cells, ؊3.9-and ؉9.5-kb regions function as transcriptional enhancers in GATA motif independent and dependent fashions, respectively. We also found that knockdown of GATA3 by RNA interference induces GATA2 in undifferentiated trophoblast cells. Interestingly, three other known GATA motif-dependent Gata2 regulatory elements, the ؊1.8-, ؊2.8-, and ؊77-kb regions, which are important to regulate Gata2 in hematopoietic cells are not occupied by GATA factors in trophoblast cells. These elements do not show any enhancer activity and also possess inaccessible chromatin structure in trophoblast cells indicating a contextdependent function. Our results indicate that GATA3 directly represses Gata2 in undifferentiated trophoblast cells, and a switch in chromatin occupancy between GATA3 and GATA2 (GATA3/GATA2 switch) induces transcription during trophoblast differentiation. We predict that this GATA3/GATA2 switch is an important mechanism for the transcriptional regulation of other trophoblast-specific genes.In the early mouse embryo, trophoectoderm overlaying the inner cell mass contains trophoblast stem (TS) 2 cells (1). During development, TS cells give rise to distinct highly differentiated trophoblast subtypes, which build the functional units of the organ, the placenta (2). Trophoblast cells are important for the anchorage of the embryo to the mother, for establishing a vascular connection for nutrient and gas transport to the embryo, and expression of hormones that are required for the successful progression of pregnancy (3). In rodents, multiple differentiated cell types can be derived from TS cells: trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast, glycogen trophoblast cells, and invasive trophoblasts (2, 4). Trophoblast giant cells are characterized by endoreduplication and expression of members of the prolactin gene family. During pregnancy, these cells invade into the uterus and promote local and systemic adaptations in the mother that are necessary for embryonic growth and survival (2, 3). Differentiation of trophoblast giant cells occurs in a spatially and temporally highly organized manner and multiple transcription factors, including GATA2 and GATA3, have been implicated in the transcriptional regulation of trophoblast giant cell-specific gene expression (5-8).The GATA family of transcription factors, GAT...