Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/β-Catenin Signaling

SummaryWnt/β-catenin signal transduction requires direct binding of β-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. β-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-β-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/β-catenin in breast cancer

Engineering an Escherichia coli strain for production of long single-stranded DNA

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA

Opposing effects of Tcf3 and Tcf1 control Wnt stimulation of embryonic stem cell self-renewal

The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on embryonic stem cell (ESC) chromatin indicated that Tcf3 has been suggested to play an integral role in a poorly understood mechanism underlying Wnt-dependent stimulation of mouse ESC self-renewal of mouse ESCs. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signalling suggested Tcf3–β-catenin activation of target genes would stimulate self-renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3 inhibition for ESC self-renewal, demonstrating that inhibition of Tcf3 repressor is the necessary downstream effect of Wnt signalling. Interestingly, both Tcf3–β-catenin and Tcf1–β-catenin interactions contributed to Wnt stimulation of self-renewal and gene expression, and the combination of Tcf3 and Tcf1 recruited Wnt-stabilized β-catenin to Oct4 binding sites on ESC chromatin. This work elucidates the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.Chemistry and Chemical Biolog

Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering.

As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo

MpzR98C arrests Schwann cell development in a mouse model of early-onset Charcot-Marie-Tooth disease type 1B

Mutations in myelin protein zero (MPZ) cause Charcot–Marie–Tooth disease type 1B. Many dominant MPZ mutations, including R98C, present as infantile onset dysmyelinating neuropathies. We have generated an R98C ‘knock-in’ mouse model of Charcot–Marie–Tooth type 1B, where a mutation encoding R98C was targeted to the mouse Mpz gene. Both heterozygous (R98C/+) and homozygous (R98C/R98C) mice develop weakness, abnormal nerve conduction velocities and morphologically abnormal myelin; R98C/R98C mice are more severely affected. MpzR98C is retained in the endoplasmic reticulum of Schwann cells and provokes a transitory, canonical unfolded protein response. Ablation of Chop, a mediator of the protein kinase RNA-like endoplasmic reticulum kinase unfolded protein response pathway restores compound muscle action potential amplitudes of R98C/+ mice but does not alter the reduced conduction velocities, reduced axonal diameters or clinical behaviour of these animals. R98C/R98C Schwann cells are developmentally arrested in the promyelinating stage, whereas development is delayed in R98C/+ mice. The proportion of cells expressing c-Jun, an inhibitor of myelination, is elevated in mutant nerves, whereas the proportion of cells expressing the promyelinating transcription factor Krox-20 is decreased, particularly in R98C/R98C mice. Our results provide a potential link between the accumulation of MpzR98C in the endoplasmic reticulum and a developmental delay in myelination. These mice provide a model by which we can begin to understand the early onset dysmyelination seen in patients with R98C and similar mutations

Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/b-Catenin Signaling

Wnt/β-catenin signal transduction requires direct binding of β-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. β-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-β-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/β-catenin in breast cancer

Regulation of Tcf7l1 DNA Binding and Protein Stability as Principal Mechanisms of Wnt/b-Catenin Signaling

Wnt/beta-catenin signal transduction requires direct binding of beta-catenin to Tcf/Lef proteins, an event that is classically associated with stimulating transcription by recruiting coactivators. This molecular cascade plays critical roles throughout embryonic development and normal postnatal life by affecting stem cell characteristics and tumor formation. Here, we show that this pathway utilizes a fundamentally different mechanism to regulate Tcf7l1 (formerly named Tcf3) activity. beta-catenin inactivates Tcf7l1 without a switch to a coactivator complex by removing it from DNA, which leads to Tcf7l1 protein degradation. Mouse genetic experiments demonstrate that Tcf7l1 inactivation is the only required effect of the Tcf7l1-beta-catenin interaction. Given the expression of Tcf7l1 in pluripotent embryonic and adult stem cells, as well as in poorly differentiated breast cancer, these findings provide mechanistic insights into the regulation of pluripotency and the role of Wnt/beta-catenin in breast cancer