Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

Abstract

SUMMARYTargeted protein degradation methods offer a unique avenue to assess a protein’s function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBPF36V tag fused to Negative elongation factor b (Nelfb) locus, a ubiquitously expressed protein regulator of transcription. In the first validation of targeted endogenous protein degradation across mammalian development, we demonstrate that irrespective of the route of administration the dTAG system is safe, rapid, and efficient in embryos from the zygote to midgestation stages. Additionally, acute early depletion of NELFB revealed a specific role in zygote-to-2-cell development and Zygotic Genome Activation (ZGA).HIGHLIGHTSgenetically engineered mouse model harboring a FKBPF36V knock-in to evaluate kinetics and efficacy of the dTAG degron system in vivosystem is non-toxic, and allows acute and efficient degradation of a FKBPF36V- tagged endogenous protein during in utero embryo developmentsystem nables fine temporal degradation and reversibility of depletion across embryonic stagesstage-specific depletion reveals a role for NELFB during mouse ZGA

RNA polymerase II and PARP1 shape enhancer-promoter contacts

“…(A) Illustration of the NELFB-dTAG system and the corresponding effect on the NELF complex. Following NELFB depletion with dTAG, the NELF complex dissociates and is no longer found bound to chromatin ( 93 ) (B) Heatmaps showing PRO-seq signal in untreated (control) mESCs (left), the fold-change in PRO-seq signal following 30 minutes of dTAG treatment and after 60 minutes of dTAG treatment near TSSs as well as the fold change in PRO-seq signal at 30 minutes and 60 minutes of dTAG treatment compared to untreated control and at 60 minutes compared to 30 minutes of dTAG treatment. (C) A line plot showing the median enhancer-promoter contacts over background ratio change relative to the untreated (T=0) control.…”

“…PRO-seq data ( 30, 33 ) for Jurkat T-cells were downloaded from GSE66031 and for K562 from GSE60455. PRO-seq data for mECSs harboring a homozygous endogenous NELFB-FKBP12 F36V fusion protein, treated and untreated with dTAG-13 ( 93 ), were downloaded from GSE196653. GRO-seq data for mESCs ( 47, 86 ) were downloaded from GSE43390 and GSE48895.…”

RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

“…Molecularly, profiling of Pol II pausing across pre-implantation mouse development has identified a distinct reduction in Pol II pausing following Zygotic Genome Activation (ZGA) until late blastocyst stage, at which point it is re-established (Liu et al, 2020). We recently showed that NELF is required at this specific stage prior to ZGA to regulate the major ZGA wave in mouse embryos (Abuhashem and Hadjantonakis 2021). At the cellular level, several studies investigating tissue-specific Nelfb knockouts have revealed that functional defects are observed when Nelfb -/- tissues are challenged by an external stimulus, such as an injury or an infection or the need to regenerate in the context of muscle stem cells, the uterine and intestinal walls, and in macrophages (Hewitt et al 2019; Ou et al 2021; Robinson et al 2021; Yu et al 2020).…”

Preimplantation embryo gene expression: 56 years of discovery, and counting

“…It is likely that a detailed understanding of the dynamic regulation of oocyte and preimplantation proteomes and functions of individual proteins will be facilitated through a combination of genetic modifications to allow tagging of proteins for in situ detection, spatial imaging, and monitoring, and to allow experimentally induced degradation and replenishment of proteins in a controlled manner. Recent technologies allowing for insertion of tags through homology‐mediated DNA editing provide such opportunities (Abuhashem & Hadjantonakis, 2021; Gu, 2020).…”