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33


Members of an array of zinc-finger proteins specify distinct Hox chromatin boundaries

Ortabozkoyun, Havva; Huang, Pin-Yao; Gonzalez-Buendia, Edgar; Cho, Hyein; Kim, Sang Y; Tsirigos, Aristotelis; Mazzoni, Esteban O; Reinberg, Danny
Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.
PMID: 39173638
ISSN: 1097-4164
CID: 5681022

T4 DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR editing

Yang, Qiaoyan; Abebe, Jonathan S; Mai, Michelle; Rudy, Gabriella; Kim, Sang Y; Devinsky, Orrin; Long, Chengzu
Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.
PMCID:11377749
PMID: 39039289
ISSN: 1460-2075
CID: 5687292

On the genetic basis of tail-loss evolution in humans and apes

Xia, Bo; Zhang, Weimin; Zhao, Guisheng; Zhang, Xinru; Bai, Jiangshan; Brosh, Ran; Wudzinska, Aleksandra; Huang, Emily; Ashe, Hannah; Ellis, Gwen; Pour, Maayan; Zhao, Yu; Coelho, Camila; Zhu, Yinan; Miller, Alexander; Dasen, Jeremy S; Maurano, Matthew T; Kim, Sang Y; Boeke, Jef D; Yanai, Itai
The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.
PMCID:10901737
PMID: 38418917
ISSN: 1476-4687
CID: 5686892

Mouse genome rewriting and tailoring of three important disease loci

Zhang, Weimin; Golynker, Ilona; Brosh, Ran; Fajardo, Alvaro; Zhu, Yinan; Wudzinska, Aleksandra M; Ordoñez, Raquel; Ribeiro-Dos-Santos, André M; Carrau, Lucia; Damani-Yokota, Payal; Yeung, Stephen T; Khairallah, Camille; Vela Gartner, Antonio; Chalhoub, Noor; Huang, Emily; Ashe, Hannah J; Khanna, Kamal M; Maurano, Matthew T; Kim, Sang Yong; tenOever, Benjamin R; Boeke, Jef D
Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.
PMCID:10632133
PMID: 37914927
ISSN: 1476-4687
CID: 5606842

A conditional counterselectable Piga knockout in mouse embryonic stem cells for advanced genome writing applications

Zhang, Weimin; Brosh, Ran; McCulloch, Laura H; Zhu, Yinan; Ashe, Hannah; Ellis, Gwen; Camellato, Brendan R; Kim, Sang Yong; Maurano, Matthew T; Boeke, Jef D
Overwriting counterselectable markers is an efficient strategy for removing wild-type DNA or replacing it with payload DNA of interest. Currently, one bottleneck of efficient genome engineering in mammals is the shortage of counterselectable (negative selection) markers that work robustly without affecting organismal developmental potential. Here, we report a conditional Piga knockout strategy that enables efficient proaerolysin-based counterselection in mouse embryonic stem cells. The conditional Piga knockout cells show similar proaerolysin resistance as full (non-conditional) Piga deletion cells, which enables the use of a PIGA transgene as a counterselectable marker for genome engineering purposes. Native Piga function is readily restored in conditional Piga knockout cells to facilitate subsequent mouse development. We also demonstrate the generality of our strategy by engineering a conditional knockout of endogenous Hprt. Taken together, our work provides a new tool for advanced mouse genome writing and mouse model establishment.
PMCID:9184564
PMID: 35692632
ISSN: 2589-0042
CID: 5282452

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

Analysis of urgent/emergent conversions from monitored anesthesia care to general anesthesia with airway instrumentation

Kim, Sang; Chang, Brian A; Rahman, Amreen; Lin, Hung-Mo; DeMaria, Samuel; Zerillo, Jeron; Wax, David B
BACKGROUND:Monitored Anesthesia Care (MAC) is an anesthetic service involving the titration of sedatives/analgesics to achieve varying levels of sedation while avoiding general anesthesia (GA) and airway instrumentation. The goal of our study was to determine the overall incidence of conversion from MAC to general anesthesia with airway instrumentation and elucidate reasons and risk factors for conversion. METHODS:In this retrospective observational study, all non-obstetric adult patients who received MAC from July 2002 to July 2015 at Mount Sinai Hospital were electronically screened for inclusion via a clinical database. Patient, procedure, anesthetic, and practitioner data were all collected and analyzed to generate descriptive analyses. Subsequent univariate and multivariate analyses were used to identify specific risk factors associated with conversion to GA. RESULTS:Overall, 0.50% (1097/219,061) of MAC cases were converted to GA. Approximately half of conversions were due to the patient's "intolerance" of MAC (with or without failed regional anesthesia), while the other half were due to physiologic derangements. Body mass index, male sex, American Society of Anesthesiologists Physical Status Classification, anesthesia team composition, and surgical specialty were all associated with risk of conversion to GA. CONCLUSIONS:This is one of the first and largest retrospective studies aimed at identifying reasons and risk factors associated with the conversion of MAC to GA. These findings may be used to help better anticipate or prevent these events.
PMCID:8240303
PMID: 34187367
ISSN: 1471-2253
CID: 5534122

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