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Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes

Kinisu, Martin; Choi, Yong Jin; Cattoglio, Claudia; Liu, Ke; Roux de Bezieux, Hector; Valbuena, Raeline; Pum, Nicole; Dudoit, Sandrine; Huang, Haiyan; Xuan, Zhenyu; Kim, Sang Yong; He, Lin
Early blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here we identify three major two-cell-stage (2C)-specific endogenous retroviruses (ERVs) as the molecular hallmark of this bi-potential plasticity. Using the long terminal repeats (LTRs) of all three 2C-specific ERVs, we identify Krüppel-like factor 5 (Klf5) as their major upstream regulator. Klf5 is essential for bi-potential cell fate; a single Klf5-overexpressing embryonic stem cell (ESC) generates terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces inner cell mass (ICM) and trophectoderm (TE) specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. The 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.
PMID: 34758315
ISSN: 2211-1247
CID: 5050592

Genetic variation of staphylococcal LukAB toxin determines receptor tropism

Perelman, Sofya S; James, David B A; Boguslawski, Kristina M; Nelson, Chase W; Ilmain, Juliana K; Zwack, Erin E; Prescott, Rachel A; Mohamed, Adil; Tam, Kayan; Chan, Rita; Narechania, Apurva; Pawline, Miranda B; Vozhilla, Nikollaq; Moustafa, Ahmed M; Kim, Sang Y; Dittmann, Meike; Ekiert, Damian C; Bhabha, Gira; Shopsin, Bo; Planet, Paul J; Koralov, Sergei B; Torres, Victor J
Staphylococcus aureus has evolved into diverse lineages, known as clonal complexes (CCs), which exhibit differences in the coding sequences of core virulence factors. Whether these alterations affect functionality is poorly understood. Here, we studied the highly polymorphic pore-forming toxin LukAB. We discovered that the LukAB toxin variants produced by S. aureus CC30 and CC45 kill human phagocytes regardless of whether CD11b, the previously established LukAB receptor, is present, and instead target the human hydrogen voltage-gated channel 1 (HVCN1). Biochemical studies identified the domain within human HVCN1 that drives LukAB species specificity, enabling the generation of humanized HVCN1 mice with enhanced susceptibility to CC30 LukAB and to bloodstream infection caused by CC30 S. aureus strains. Together, this work advances our understanding of an important S. aureus toxin and underscores the importance of considering genetic variation in characterizing virulence factors and understanding the tug of war between pathogens and the host.
PMID: 33875847
ISSN: 2058-5276
CID: 4846982

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency

Vidal, Simon E; Polyzos, Alexander; Chatterjee, Kaushiki; Ee, Ly-Sha; Swanzey, Emily; Morales-Valencia, Jorge; Wang, Hongsu; Parikh, Chaitanya N; Amlani, Bhishma; Tu, Shengjiang; Gong, Yixiao; Snetkova, Valentina; Skok, Jane A; Tsirigos, Aristotelis; Kim, Sangyong; Apostolou, Effie; Stadtfeld, Matthias
Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.
PMID: 32976761
ISSN: 2213-6711
CID: 4606132

Physiological expression and function of the MDR1 transporter in cytotoxic T lymphocytes

Chen, Mei Lan; Sun, Amy; Cao, Wei; Eliason, Amber; Mendez, Kayla M; Getzler, Adam J; Tsuda, Shanel; Diao, Huitian; Mukori, Clever; Bruno, Nelson E; Kim, Sang Yong; Pipkin, Matthew E; Koralov, Sergei B; Sundrud, Mark S
Multidrug resistance-1 (MDR1) acts as a chemotherapeutic drug efflux pump in tumor cells, although its physiological functions remain enigmatic. Using a recently developed MDR1-knockin reporter allele (Abcb1aAME), we found that constitutive MDR1 expression among hematopoietic cells was observed in cytolytic lymphocytes-including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer cells-and regulated by Runt-related (Runx) transcription factors. Whereas MDR1 was dispensable for naive CD8+ T cell development, it was required for both the normal accumulation of effector CTLs following acute viral infection and the protective function of memory CTLs following challenge with an intracellular bacterium. MDR1 acted early after naive CD8+ T cell activation to suppress oxidative stress, enforce survival, and safeguard mitochondrial function in nascent CTLs. These data highlight an important endogenous function of MDR1 in cell-mediated immune responses and suggest that ongoing efforts to intentionally inhibit MDR1 in cancer patients could be counterproductive.
PMID: 32302378
ISSN: 1540-9538
CID: 4383912

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids

Cao, Wei; Kayama, Hisako; Chen, Mei Lan; Delmas, Amber; Sun, Amy; Kim, Sang Yong; Rangarajan, Erumbi S; McKevitt, Kelly; Beck, Amanda P; Jackson, Cody B; Crynen, Gogce; Oikonomopoulos, Angelos; Lacey, Precious N; Martinez, Gustavo J; Izard, Tina; Lorenz, Robin G; Rodriguez-Palacios, Alex; Cominelli, Fabio; Abreu, Maria T; Hommes, Daniel W; Koralov, Sergei B; Takeda, Kiyoshi; Sundrud, Mark S
PMID: 32187521
ISSN: 1097-4180
CID: 4352762

Exploiting species specificity to understand the tropism of a human-specific toxin

Boguslawski, K M; McKeown, A N; Day, C J; Lacey, K A; Tam, K; Vozhilla, N; Kim, S Y; Jennings, M P; Koralov, S B; Elde, N C; Torres, V J
Many pathogens produce virulence factors that are specific toward their natural host. Clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) isolates are highly adapted to humans and produce an array of human-specific virulence factors. One such factor is LukAB, a recently identified pore-forming toxin that targets human phagocytes by binding to the integrin component CD11b. LukAB exhibits strong tropism toward human, but not murine, CD11b. Here, phylogenetics and biochemical studies lead to the identification of an 11-residue domain required for the specificity of LukAB toward human CD11b, which is sufficient to render murine CD11b compatible with toxin binding. CRISPR-mediated gene editing was used to replace this domain, resulting in a "humanized" mouse. In vivo studies revealed that the humanized mice exhibit enhanced susceptibility to MRSA bloodstream infection, a phenotype mediated by LukAB. Thus, these studies establish LukAB as an important toxin for MRSA bacteremia and describe a new mouse model to study MRSA pathobiology.
PMID: 32195339
ISSN: 2375-2548
CID: 4353762

Widespread Transcriptional Scanning in the Testis Modulates Gene Evolution Rates

Xia, Bo; Yan, Yun; Baron, Maayan; Wagner, Florian; Barkley, Dalia; Chiodin, Marta; Kim, Sang Y; Keefe, David L; Alukal, Joseph P; Boeke, Jef D; Yanai, Itai
The testis expresses the largest number of genes of any mammalian organ, a finding that has long puzzled molecular biologists. Our single-cell transcriptomic data of human and mouse spermatogenesis provide evidence that this widespread transcription maintains DNA sequence integrity in the male germline by correcting DNA damage through a mechanism we term transcriptional scanning. We find that genes expressed during spermatogenesis display lower mutation rates on the transcribed strand and have low diversity in the population. Moreover, this effect is fine-tuned by the level of gene expression during spermatogenesis. The unexpressed genes, which in our model do not benefit from transcriptional scanning, diverge faster over evolutionary timescales and are enriched for sensory and immune-defense functions. Collectively, we propose that transcriptional scanning shapes germline mutation signatures and modulates mutation rates in a gene-specific manner, maintaining DNA sequence integrity for the bulk of genes but allowing for faster evolution in a specific subset.
PMID: 31978344
ISSN: 1097-4172
CID: 4273592

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice

Lubkin, Ashira; Lee, Warren L; Alonzo, Francis; Wang, Changsen; Aligo, Jason; Keller, Matthew; Girgis, Natasha M; Reyes-Robles, Tamara; Chan, Rita; O'Malley, Aidan; Buckley, Peter; Vozhilla, Nikollaq; Vasquez, Marilyn T; Su, Johnny; Sugiyama, Michael; Yeung, Stephen T; Coffre, Maryaline; Bajwa, Sofia; Chen, Eric; Martin, Patricia; Kim, Sang Y; Loomis, Cynthia; Worthen, G Scott; Shopsin, Bo; Khanna, Kamal M; Weinstock, Daniel; Lynch, Anthony Simon; Koralov, Sergei B; Loke, P'ng; Cadwell, Ken; Torres, Victor J
The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.
PMID: 30799265
ISSN: 1934-6069
CID: 3721612

Uroplakins play conserved roles in egg fertilization and acquired additional urothelial functions during mammalian divergence

Liao, Yi; Chang, Hung-Chi; Liang, Feng-Xia; Chung, Pei-Jung; Wei, Yuan; Nguyen, Tuan-Phi; Zhou, Ge; Talebian, Sheeva; Krey, Lewis C; Deng, Fang-Ming; Wong, Tak-Wah; Chicote, Javier U; Grifo, James A; Keefe, David L; Shapiro, Ellen; Lepor, Herbert; Wu, Xue-Ru; DeSalle, Robert; Garcia-España, Antonio; Kim, Sang Yong; Sun, Tung-Tien
Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique 2D-crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mouse urothelium and are often referred to as being urothelium-specific, they are also expressed in several nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms and ovary/oocytes. In oocytes, uroplakins co-localize with CD9 on cell surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine-phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require 2D-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form 2D- crystalline plaques during mammalian divergence enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, in order to protect/modify the apical surface of the modern-day mammalian urothelium.
PMID: 30303751
ISSN: 1939-4586
CID: 3335002

Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes

Amlani, Bhishma; Liu, Yiyuan; Chen, Taotao; Ee, Ly-Sha; Lopez, Peter; Heguy, Adriana; Apostolou, Effie; Kim, Sang Yong; Stadtfeld, Matthias
The ability of induced pluripotent stem cells (iPSCs) to differentiate into all adult cell types makes them attractive for research and regenerative medicine; however, it remains unknown when and how this capacity is established. We characterized the acquisition of developmental pluripotency in a suitable reprogramming system to show that iPSCs prior to passaging become capable of generating all tissues upon injection into preimplantation embryos. The developmental potential of nascent iPSCs is comparable to or even surpasses that of established pluripotent cells. Further functional assays and genome-wide molecular analyses suggest that cells acquiring developmental pluripotency exhibit a unique combination of properties that distinguish them from canonical naive and primed pluripotency states. These include reduced clonal self-renewal potential and the elevated expression of differentiation-associated transcriptional regulators. Our observations close a gap in the understanding of induced pluripotency and provide an improved roadmap of cellular reprogramming with ramifications for the use of iPSCs.
PMID: 29420174
ISSN: 2211-1247
CID: 2947822