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1.
J Invest Dermatol ; 144(5): 936-949, 2024 May.
Article in English | MEDLINE | ID: mdl-38643988

ABSTRACT

The epidermis is the body's first line of protection against dehydration and pathogens, continually regenerating the outermost protective skin layers throughout life. During both embryonic development and wound healing, epidermal stem and progenitor cells must respond to external stimuli and insults to build, maintain, and repair the cutaneous barrier. Recent advances in CRISPR-based methods for cell lineage tracing have remarkably expanded the potential for experiments that track stem and progenitor cell proliferation and differentiation over the course of tissue and even organismal development. Additional tools for DNA-based recording of cellular signaling cues promise to deepen our understanding of the mechanisms driving normal skin morphogenesis and response to stressors as well as the dysregulation of cell proliferation and differentiation in skin diseases and cancer. In this review, we highlight cutting-edge methods for cell lineage tracing, including in organoids and model organisms, and explore how cutaneous biology researchers might leverage these techniques to elucidate the developmental programs that support the regenerative capacity and plasticity of the skin.


Subject(s)
Cell Differentiation , Cell Lineage , Humans , Animals , Skin/cytology , Stem Cells/cytology , Cell Proliferation , Regeneration/physiology
2.
Cell ; 187(10): 2411-2427.e25, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38608704

ABSTRACT

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.


Subject(s)
CRISPR-Cas Systems , Chromatin , Epigenesis, Genetic , Gene Editing , Humans , Chromatin/metabolism , Chromatin/genetics , CRISPR-Cas Systems/genetics , Gene Editing/methods , Histones/metabolism , Transcription Factors/metabolism , Histone Code
3.
bioRxiv ; 2023 Apr 12.
Article in English | MEDLINE | ID: mdl-37090511

ABSTRACT

Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis- chromatin environment on prime editing efficiency. Using a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated "sensor", we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans -acting factors with the cis -chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis -chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. altering chromatin state in a locus-specific manner in order to increase or decrease the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful.

4.
Sci Adv ; 7(3)2021 01.
Article in English | MEDLINE | ID: mdl-33523889

ABSTRACT

The G1-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G1-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G1-S transition, cells triggering the DNA damage checkpoint in G1 phase route back to quiescence, and this cellular rerouting can be initiated at any point in G1 phase. Furthermore, we find that most of the cells receiving damage in G1 phase actually fail to arrest and proceed through the G1-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G1 phase and indicate that cells have a G1 checkpoint.


Subject(s)
DNA Damage , Cell Cycle/genetics , Cell Division , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , S Phase
5.
Nat Commun ; 11(1): 24, 2020 01 07.
Article in English | MEDLINE | ID: mdl-31911655

ABSTRACT

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.


Subject(s)
Anaphase , Cell Cycle Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Metaphase , Poly-ADP-Ribose Binding Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Cycle Proteins/genetics , Cell Line , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mitosis , Poly-ADP-Ribose Binding Proteins/genetics , Promyelocytic Leukemia Protein/genetics , Promyelocytic Leukemia Protein/metabolism , Protein Binding , Proteolysis , Retinoblastoma-Binding Protein 7/genetics , Retinoblastoma-Binding Protein 7/metabolism , Spindle Apparatus/metabolism , Ubiquitin-Protein Ligases/genetics
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