Mapping the chromatin landscape and Blimp1 transcriptional targets that regulate trophoblast differentiation

Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-β gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades

Single-cell RNA-seq reveals cell type-specific transcriptional signatures at the maternal-foetal interface during pregnancy

Growth and survival of the mammalian embryo within the uterine environment depends on the placenta, a highly complex vascularized organ comprised of both maternal and foetal tissues. Recent experiments demonstrate that the zinc finger transcriptional repressor Prdm1/Blimp1 is essential for specification of spiral artery trophoblast giant cells (SpA-TGCs) that invade and remodel maternal blood vessels. To learn more about functional contributions made by Blimp1+ cell lineages here we perform the first single-cell RNA-seq analysis of the placenta. Cell types of both foetal and maternal origin are profiled. Comparisons with microarray datasets from mutant placenta and in vitro differentiated trophoblast stem cells allow us to identify Blimp1-dependent transcripts enriched in SpA-TGCs. Our experiments provide new insights into the functionally distinct cell types present at the maternal–foetal interface and advance our knowledge of dynamic gene expression patterns controlling placental morphogenesis and vascular mimicry

Blimp1/Prdm1 functions in opposition to Irf1 to maintain neonatal tolerance during postnatal intestinal maturation

The neonatal intestine is a very complex and dynamic organ that must rapidly adapt and remodel in response to a barrage of environmental stimuli during the first few postnatal weeks. Recent studies demonstrate that the zinc finger transcriptional repressor Blimp1/Prdm1 plays an essential role governing postnatal reprogramming of intestinal enterocytes during this period. Functional loss results in global changes in gene expression patterns, particularly in genes associated with metabolic function. Here we engineered a knock-in allele expressing an eGFP-tagged fusion protein under control of the endogenous regulatory elements and performed genome wide ChIP-seq analysis to identify direct Blimp1 targets and further elucidate the function of Blimp1 in intestinal development. Comparison with published human and mouse datasets revealed a highly conserved core set of genes including interferon-inducible promoters. Here we show that the interferon-inducible transcriptional activator Irf1 is constitutively expressed throughout fetal and postnatal intestinal epithelium development. ChIP-seq demonstrates closely overlapping Blimp1 and Irf1 peaks at key components of the MHC class I pathway in fetal enterocytes. The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition. Collectively, these experiments strongly suggest that in addition to regulating the enterocyte metabolic switch, Blimp1 functions as a gatekeeper in opposition to Irf1 to prevent premature activation of the MHC class I pathway in villus epithelium to maintain tolerance in the neonatal intestine

Dual Loss of Rb1 and Trp53 in the Adrenal Medulla Leads to Spontaneous Pheochromocytoma1

Using a Cre/loxP system, we have determined the phenotypic consequences attributable to in vivo deletion of both Rb1 and Trp53 in the mouse adrenal medulla. The coablation of these two tumor suppressor genes during embryogenesis did not disrupt adrenal gland development but resulted in the neoplastic transformation of the neural crest-derived adrenal medulla, yielding pheochromocytomas (PCCs) that developed with complete penetrance and were inevitably bilateral. Despite their typically benign status, these PCCs had profound ramifications on mouse vitality, with effected mice having a median survival of only 121 days. Evaluation of these PCCs by both immunohistochemistry and electron microscopy revealed that most Rb1-/-:Trp53-/- chromaffin cells possessed atypical chromagenic vesicles that did not seem capable of appropriately storing synthesized catecholamines. The structural remodeling of the heart in mice harboring Rb1-/-:Trp53-/- PCCs suggests that the mortality of these mice may be attributable to the inappropriate release of catecholamines from the mutated adrenal chromaffin cells. On the basis of the collective data from Rb1 and Trp53 knockout mouse models, it seems that the conversion of Rb1 loss-driven adrenal medulla hyperplasia to PCC can be greatly enhanced by the compound loss of Trp53, whereas the loss of Trp53 alone is generally ineffectual on adrenal chromaffin cell homeostasis. Consequently, the Trp53 tumor suppressor gene is an efficient genetic modifier of Rb1 loss in the development of PCC, and their compound loss in the adrenal medulla has a profound impact on both cellular homeostasis and animal vitality

The eGFP-tagged Blimp1-fusion protein is efficiently expressed in the developing placenta and embryonic small intestine, but fails to rescue germ cell defects.

<p>(A) Immunohistochemical staining of E9.5 placentae demonstrates the eGFP-tagged Blimp1-fusion protein is correctly expressed in the spongiotrophoblast layer. The position of the central maternal artery is outlined. Bar, 200 μm. SpT, spongiotrophoblast; dec, maternal decidua; lab, labyrinth trophoblast. (B) Western blot analysis demonstrates robust expression in E16.5 small intestine. RIPA lysates of STO fibroblasts are included as a negative control. Levels of Blimp1 expression were quantified relative to the first wild type littermate. (C) Immunohistochemical staining demonstrates nuclear expression of both endogenous and eGFP-tagged Blimp1 protein in E16.5 villus epithelium. Bar, 200 μm. (D) The eGFP-tagged Blimp1 knock-in allele fails to reconstitute germ cell formation. Fast red alkaline phosphatase staining at E7.5 demonstrates markedly reduced numbers of PGCs (black arrows). Hematoxylin- and eosin-stained sections confirm that adult homozygous BEG/BEG testes lack spermatocytes. Bar, 200 μm.</p

The eGFP-tagged Blimp1 knock-in allele efficiently rescues plasma cell differentiation.

<p>(A) Schematic representation of the targeting vector, wild type locus, and Southern blot screening strategy. (B) Intercross matings of heterozygous BEG animals generate Mendelian ratios of wild type, heterozygous and homozygous mutant progeny. (C) Western blot analysis demonstrates the eGFP-Blimp1 fusion protein is robustly induced in LPS-treated splenocytes. Levels of Blimp1 protein were quantified relative to the 129/SvEv wild type sample. Additional positive controls were wild type C57BL/6 and a BEG intercross littermate (+/+), heterozygous Blimp1-Venus BAC transgene (Blimp1-Venus) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref025" target="_blank">25</a>] or Blimp1-IRES-GFP (Blimp1-GFP/+) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref026" target="_blank">26</a>] reporter strains. Prdm1ΔEx1A splenocytes lacking the ability to generate plasma cells were included as a negative control [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005375#pgen.1005375.ref027" target="_blank">27</a>]. (D) Imagestream analysis of LPS-treated splenocytes reveals nuclear localization of the Blimp1-eGFP fusion protein in contrast to the cytoplasmic/plasma membrane localization of Venus expressed under the control of the Blimp1 BAC-transgene. (E) Homozygous mutant B cells that exclusively express the eGFP-tagged Blimp1-fusion protein efficiently undergo plasma cell (CD138+B220+) terminal differentiation.</p

BLIMP1 and IRF1 are co-expressed in fetal human intestine.

<p>Nuclear BLIMP1 staining detected in the pseudostratified epithelium of the developing gut tube at (A) 9 weeks of gestation persists in (B) the villus epithelium including the immature crypts of Lieberkuhn at 15 weeks of gestation. (C) Although not detected in the gut tube prior to villus formation nuclear IRF1 staining is present in the villus epithelium at (D) 15 weeks of gestation. (A, C) Bar, 800 μm, (B, D) Bar 200 μm.</p