Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation.
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ABSTRACT:
PROVIDER: PRJNA490158 | ENA |
REPOSITORIES: ENA
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Project description:The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.
2020-01-16 | GSE119737 | GEO
Project description:Wnt/b-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce b-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with an ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves casein kinase-1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing b-catenin turnover and propelling ligandindependent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.
2020-08-14 | PXD020478 | Pride
Project description:Acute Myeloid Leukemia (AML) is a blood cancer complicated by acquired drug resistance, disease relapse, and low overall survival rates. Combination therapies using multiple targeted inhibitors can effectively treat AML patients. However, combination treatments are limited by the number of useable targets and our ability to create rational pairings using complimentary molecular mechanisms. Here, we used a human kinase domain-targeted CRISPR screen to identify histone H3 associated protein kinase (HASPIN) as a significant, understudied dependency in AML. HASPIN depletion significantly reduced growth rate, induced a cell cycle arrest, and dysregulated transcription in AML. A proteomics datamining study characterized splicing factors as major HASPIN kinase substrates and highlighted HASPIN’s role as a splicing regulatory kinase. Accordingly, HASPIN depletion strongly dysregulated splicing and inversed aberrant, pro-leukemic splicing programs in AML patients. HASPIN inhibitor CHR-6494 effectively reduced cell viability across AML subtypes while sparing healthy cells. Furthermore, a novel combination therapy consisting of CHR-6494 and BCL-2 inhibitor Venetoclax synergistically reduced AML cell viability and resensitized Venetoclax-resistant AMLs to treatment. Our study presents HASPIN kinase as a novel, therapeutic target for AML, underscores an underappreciated role in splicing regulation, and proposes a viable combination treatment for clinical use.
2025-08-04 | GSE281301 | GEO
Project description:The atypical protein kinase Haspin phosphorylates Histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph creates a docking site for the Chromosomal Passenger Complex at the inner centromere, enabling correction of erratic microtubule-chromosome contacts and preventing chromosome mis-segregation. Surprisingly, Haspin knockout in the mouse does not cause developmental defects, except for testicular anomalies. Therefore, the physiological role of this kinase and the mechanistic significance of H3T3ph are in question. We show here that mouse embryonic stem cells that lack or overexpress Haspin are prone to chromosome misalignment. However, the cell population as a whole maintains the ability to expand and differentiate into multiple lineages. Although not essential for pluripotency, Haspin affects the expression of several testis-specific genes. Furthermore, the H3T3ph mark is detected in under-condensed chromatin domains of haploid spermatids, in cis with lysine/arginine methylation marks. We propose that Haspin controls a compound phospho-methyl switch and regulates testis-specific transcription.
2021-10-06 | GSE154246 | GEO
Project description:The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.
| S-EPMC6959359 | biostudies-literature
Project description:Although histone-modifying enzymes are assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here we show the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT–deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT–independent role of Tip60 in ESC maintenance. In contrast, KAT–deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a second, KAT–dependent function in differentiation. Consistent with this phenotype, KAT–deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT–dependent and KAT–independent functions of Tip60 in ESCs and during embryonic development, raising the possibility of undiscovered catalysis-independent functions of additional KATs.
2017-04-06 | GSE85505 | GEO
Project description:Cell lines geneticially engineered to undergo conditional asymmetric self-renewal were used to identify genes whose expression is asymmetric self-renewal associated (ASRA). Non-random sister chromatid segregation occurs concordantly with asymmetric self-renewal in these cell lines. Asymmetric self-renewal occurs when murine embryo fibroblasts that are otherwise p53-null are induced to express physiological levels of wildtype p53 protein (Asym). To distinguish p53-responsive genes that also require induction of asymmetric self renewal (i.e., ASRA genes) and/or non-random sister chromatid segregation for change, an additional control cell line, which continues to symmetrically self-renew (with random sister chromatid segregation) even when p53 is induced, was also compared (Symp53). This congenic cell line constitutively expresses the type II inosine monophosphate dehydrogenase (IMPDH II; the rate-limiting enzmye for guanine ribonucleotide biosynthesis) and, thereby, prevents p53-induced asymmetric self-renewal and non-random sister chromatid segregation. Three biological replicates of asymmetrically self-renewing cultures (Asym1-3) were compared with cultures that were symmetrically self-renewing - either because they did not express p53 (3 biological replicates, Sym1-3) or they expressed constitutive IMPDH II (i.e., not regulated by p53) as well as p53 (2 biological replicates, Symp53_1 and 2.)
2010-12-22 | E-GEOD-25334 | biostudies-arrayexpress
Project description:SAGA and ATAC are two related transcriptional coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development but the precise role of the two complexes in RNA polymerase II transcription in mammals is less understood. To determine whether SAGA and ATAC have redundant or specific functions dependent on their HAT activities, we compared the effects of HAT inactivation in each complex with that of inactivation of either SAGA or ATAC core subunits in mouse embryonic stem cells (ESCs). We show that core subunits of SAGA or ATAC subunits are required for complex assembly, mouse ESC growth and self-renewal. Additionally, ATAC, but not SAGA subunits are required for ESC viability by regulating the transcription of translation-related genes. Surprisingly, depletion of specific or shared HAT module subunits caused a global decrease in histone H3K9 acetylation, but did not result in significant phenotypic or transcriptional defects. Thus, our results indicate that SAGA and ATAC are differentially required for viability and self-renewal of mouse ESCs by regulating transcription through different pathways, in a HAT-independent manner.
2021-08-11 | PXD027026 | Pride
Project description:Cell lines geneticially engineered to undergo conditional asymmetric self-renewal were used to identify genes whose expression is asymmetric self-renewal associated (ASRA). Non-random sister chromatid segregation occurs concordantly with asymmetric self-renewal in these cell lines. Asymmetric self-renewal occurs when murine embryo fibroblasts that are otherwise p53-null are induced to express physiological levels of wildtype p53 protein (Asym). To distinguish p53-responsive genes that also require induction of asymmetric self renewal (i.e., ASRA genes) and/or non-random sister chromatid segregation for change, an additional control cell line, which continues to symmetrically self-renew (with random sister chromatid segregation) even when p53 is induced, was also compared (Symp53). This congenic cell line constitutively expresses the type II inosine monophosphate dehydrogenase (IMPDH II; the rate-limiting enzmye for guanine ribonucleotide biosynthesis) and, thereby, prevents p53-induced asymmetric self-renewal and non-random sister chromatid segregation.
2010-12-22 | GSE25334 | GEO
Project description:Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1AΔSET); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1AΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1AΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency.
2017-09-26 | GSE98988 | GEO