Biology subjects

Kueh, A. J.

Publications and source records attributed to Kueh, A. J..

12 recordsLinked to original sources

bioRxiv2026

SMCHD1's DNA binding activity enables its stable retention on chromatin

Chromatin proteins play critical roles in gene regulation, yet frequently we do not fully understand how weak DNA binding affinity of such proteins contributes to their locus-specific actions. Here, we studied SMCHD1, a non-canonical SMC-family protein involved in three-dimensional genome organization and gene repression of the inactive X chromosome and its autosomal targets. We replaced endogenous SMCHD1 with GFP-tagged wild-type or hinge-domain DNA-binding mutant SMCHD1 to define the cellular role of DNA binding. The mutant showed reduced enrichment at the inactive X chromosome in female cells, while retaining stable binding at most autosomal binding sites. Impaired DNA binding weakens SMCHD1-mediated gene repression and chromatin-state regulation, producing hypomorphic effect. Multiple live-cell imaging methods reveal that DNA binding constrains SMCHD1 mobility and supports maintenance, rather than initial recruitment, of chromatin-bound SMCHD1 both during interphase and mitosis. Thus, SMCHD1's weak and sequence-independent DNA binding is a key determinant of its chromatin residence, localization and function. Our findings provide a framework for understanding SMCHD1 and other chromatin proteins with sequence-independent DNA binding activity.

genomics↗

bioRxiv2025

The kinase domain of RIPK3 tunes its scaffolding functions

The pro-inflammatory programmed cell death pathway, necroptosis, relies on phosphorylation of the terminal effector, MLKL, by RIPK3. RIPK3-deficient mice or those harboring the kinase-inactivating mutation, RIPK3K51A, are ostensibly normal in the absence of challenge, indicating that RIPK3 and its kinase activity are dispensable for development. However, another kinase-inactivating mutation, RIPK3D161N, results in embryonic lethality in mice due to widespread apoptosis. As a result, the RIPK3D161N mutation is thought to confer a toxic gain-of-function. Here, to further explore the impacts of RIPK3 inactivation, we compared the stability and cellular interactions of RIPK3D161N and RIPK3K51A to a third previously-uncharacterized kinase-dead variant, RIPK3D143N. Here, we show that RIPK3K51A was unstable and did not associate with RIPK1, RIPK3D161N was unstable but interacted with RIPK1, whereas RIPK3D143N was stable and bound RIPK1 in a manner comparable to wild-type RIPK3. Thus, all three variants scaffold differently, suggesting that the assembly of cell death machinery by RIPK3 is finely tuned, not just by its kinase activity, but also by the conformation of its kinase domain. Physiologically, Ripk3D143N/D143N mice exhibited a partially penetrant lethality in utero. However, once born, Ripk3D143N/D143N mice were fertile and phenotypically indistinguishable from wild-type mice in the absence of challenge, with RIPK3D143N mutation protecting mice lacking intestinal epithelial Caspase-8 from necroptotic ileitis. Our studies support the idea that RIPK3 is a nexus between apoptotic and necroptotic signaling, and highlight the importance of considering kinase domain conformation in RIPK3 inhibitor development.

biochemistry↗

bioRxiv2023

Relative importance of MCL-1's Anti-Apoptotic versus Non-Apoptotic Functions in vivo

MCL-1 is an anti-apoptotic member of the BCL-2 protein family that ensures cell survival by blocking the intrinsic apoptotic cell death pathway1. MCL-1 is unique in being essential for early embryonic development and the survival of many cell types, including many cancer cells, which are not affected by the loss of the other anti-apoptotic BCL-2 family members1-4. Non-apoptotic functions of MCL-1 controlling mitochondrial ATP production and dynamics have been proposed to underlie this unique requirement for MCL-15-9. The relative contributions of the anti-apoptotic versus the non-apoptotic functions of MCL-1 in normal physiology have not been addressed. Here we replaced the coding sequence for MCL-1 with those for the anti-apoptotic proteins BCL-XL, BCL-2 or A1. We hypothesised that BCL-XL, BCL-2 and A1 may substitute for MCL-1 in the inhibition of apoptosis, but that they will not be able to replace MCL-1s non-apoptotic function. Strikingly, Mcl-1Bcl-xL/Bcl-xL and Mcl-1Bcl-2/Bcl-2 embryos survived to embryonic day 14.5, greatly surpassing the pre-implantation lethality of Mcl-1-/- embryos at E3.5. This demonstrates that the non-apoptotic functions of MCL-1 are dispensable for early development. However, at later stages of development and life after birth many cell types, particularly ones with high energy demand, were found to require both the anti-apoptotic and the non-apoptotic functions of MCL-1. These findings reveal the relative importance of these distinct functions of MCL-1 in physiology, providing important information for basic biology and the advancement of MCL-1 inhibitors in cancer therapy.

cell biology↗

bioRxiv2022

Deletion of the transcriptional regulator TFAP4 accelerates c-MYC-driven lymphomagenesis

Many lymphoid malignancies arise from deregulated c-MYC expression in cooperation with additional genetic lesions. While many of these cooperative genetic lesions have been discovered, DNA sequence data suggest that many more do exist. However, their contributions to c-MYC driven lymphomagenesis have not yet been investigated. We identified TFAP4 as a potent suppressor of c-MYC driven lymphoma development in a previous genome-wide CRISPR knockout screen in primary cells in vivo (Mizutani et al, 2022). CRISPR deletion of TFAP4 in E-MYC transgenic hematopoietic stem and progenitor cells (HSPCs) significantly accelerated c-MYC-driven lymphoma development in mice. TFAP4 deficient E-MYC lymphomas all arose at the pre-B cell stage. Characterization of the transcriptional profile of pre-leukemic pre-B cells in E-MYC/Cas9/sgTFAP4 transplanted mice, revealed that TFAP4 deletion reduced expression of several master regulators of B cell differentiation, such as Spi1, SpiB and Pax5, which all have been shown to be bound by TFAP4. We therefore conclude that loss of TFAP4 leads to a block in differentiation during early B cell development, causing accelerated c-MYC-driven lymphoma development.

cancer biology↗

bioRxiv2022

Cleavage of cFLIP restrains cell death during viral infection and tissue injury and favors tissue repair

Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. cFLIP is a crucial regulator of cell death and a substrate of Caspase-8. Yet, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we discovered an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant, CflipD377A, exhibited increased sensitivity to SARS-CoV-induced lethality, impaired skin wound healing and increased tissue damage caused by Sharpin deficiency. In vitro, abrogation of cFLIP cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on Gln(Q)469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress, to ensure the execution of repair programs.

cell biology↗

bioRxiv2022

MLKL deficiency protects against low-grade, sterile inflammation in aged mice

MLKL and RIPK3 are the core signaling proteins of the inflammatory cell death pathway, necroptosis, which is a known mediator and modifier of human disease. Necroptosis has been implicated in the progression of disease in almost every physiological system and recent reports suggest a role for necroptosis in aging. Here we present the first comprehensive analysis of age-related histopathological and immunological phenotypes in a cohort of Mlkl-/- and Ripk3-/- mice on a congenic C57BL/6J genetic background. We show that genetic deletion of Mlkl, but not Ripk3, in female mice interrupts immune system aging, specifically delaying the age-related reduction of circulating lymphocytes. Mlkl-/- female mice were also protected against age-related, low-grade chronic sterile inflammation, with a reduced number of inflammatory infiltrates present in the connective and muscle tissue at 17 months relative to wild-type littermates. These observations implicate MLKL in age-related sterile inflammation, suggesting a possible application for long-term anti-necroptotic therapy in humans.

immunology↗

bioRxiv2022

Survey of activation-induced genome architecture reveals a novel enhancer of Myc

The transcription factor Myc is critically important in driving cell proliferation, a function that is frequently dysregulated in cancer. To avoid this dysregulation Myc is tightly controlled by numerous layers of regulation. One such layer is the use of distal regulatory enhancers to drive Myc expression. Here, using chromosome conformation capture to examine B cells of the immune system in the first hours after their activation, we reveal a previously unidentified enhancer of myc. The interactivity of this enhancer coincides with a dramatic, but discrete, spike in Myc expression 3 hours post-activation. However, genetic deletion of this region, has little impact on Myc expression, Myc protein level or in vitro and in vivo cell proliferation. Examination of the enhancer deleted regulatory landscape suggests that enhancer redundancy likely sustains Myc expression. This work highlights not only the importance of temporally examining enhancers, but also the complexity and dynamics of the regulation of critical genes such as Myc.

genetics↗

bioRxiv2022

SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine

Suppressor Of Cytokine Signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

cell biology↗

bioRxiv2022

Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC (which is abnormally highly expressed in [~]70% of human cancers) we have used a genome-wide CRISPR knockout screen in E{micro}-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discovered that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice. Low expression of the GATOR1 complex genes correlated with poor survival outcomes for human patients with high MYC- expressing cancers. Murine lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors as a single agent therapy, both in vitro and in vivo. These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition. Key PointsO_LIIn vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor of c-MYC-driven lymphomagenesis. C_LIO_LIGATOR1 deficiency in MYC-driven lymphomas confers sensitivity to mTOR inhibition, suggesting a novel therapeutic approach. C_LI

cancer biology↗

bioRxiv2021

The inflammasome-activated cytokine IL-1 is targeted for ubiquitylation and proteasomal degradation to limit its inflammatory potential

Interleukin-1{beta} (IL-1{beta}) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in wide-spread diseases. Therefore, IL-1{beta} activity must be fine-tuned to enable antimicrobial responses whilst limiting collateral damage. Here we report that precursor IL-1{beta} is rapidly turned over by the proteasome and this correlates with its decoration by K11-, K63- and K48-linked ubiquitin chains. The ubiquitylation of IL-1{beta} is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1{beta} cleavage by caspase-1. We further demonstrate that IL-1{beta} K133 is modified by ubiquitin and forms a salt bridge with IL-1{beta} D129. Loss of IL-1{beta} K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1{beta}. Accordingly, IL-1{beta}K133R/K133R mice display increased precursor IL-1{beta} upon inflammasome priming and increased bioactive IL-1{beta}, both in vitro and following LPS injection in vivo. These findings reveal new mechanisms for limiting IL-1{beta} activity and safeguarding against damaging inflammation.

immunology↗

bioRxiv2020

Severe dysregulation of TNF expression leads to multiple inflammatory diseases and embryonic death

Post-transcriptional regulation mechanisms regulate mRNA stability or translational efficiency via ribosomes and recent evidence indicates that it is a major determinant of the accurate levels of cytokine mRNAs. While transcriptional regulation of Tnf has been well studied and found to be important for the rapid induction of Tnf mRNA and regulation of the acute phase of inflammation, study of its post-transcriptional regulation has been largely limited to the role of the AU-rich element (ARE), and to a lesser extent, that of the constitutive decay element (CDE). We have identified a new regulatory element (NRE) in the 3 untranslated region (3UTR) of Tnf, and demonstrate that ARE, CDE and NRE cooperate to efficiently down regulate Tnf expression and prevent autoimmune inflammatory diseases. We also show for the first time that excessive TNF may lead to embryonic death.

immunology↗

bioRxiv2019

An Erg driven transcriptional program controls B-lymphopoiesis

Summary/AbstractB-cell development is initiated by the stepwise differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating the mature B-cells that mediate protective immunity. This highly regulated process also generates clonal immunological diversity via recombination of immunoglobulin genes. While several transcription factors that control B-cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for the earliest steps in B-cell differentiation. Erg initiates a transcriptional network involving the B-cell lineage defining genes, Ebf1 and Pax5, that directly promotes the expression of key genes involved in V(D)J recombination and formation of the B-cell receptor. Complementation of the Erg-deficiency with a productively rearranged immunoglobulin gene rescued B-cell development, demonstrating that Erg is an essential and exquisitely stage specific regulator of the gene regulatory network controlling B-lymphopoiesis.

immunology↗