Biology subjects

Kranc, K. R.

Publications and source records attributed to Kranc, K. R..

7 recordsLinked to original sources

bioRxiv2026

Cellular context restricts a promiscuous m6A reader IDR to a single functional effector interface for mRNA decay

N6-methyladenosine (m6A) is a conserved mRNA modification that regulates transcript stability, yet how m6A readers engage effectors remains unknown. We show that during yeast meiosis, the YTH-domain protein Pho92 promotes the decay of m6A-modified transcripts through an uncharacterised, promiscuous intrinsically disordered region (IDR). In vitro, the Pho92 IDR makes multiple contacts with the Ccr4-NOT deadenylase complex and drives tethered reporter decay in the absence of any single subunit. Yet in meiosis, turnover of endogenous m6A-modified transcripts strictly depends on a single interface: direct binding of the Pho92 IDR to Caf40. A single hydrophobic residue substitution is sufficient to abolish Caf40 binding and halt transcript decay. Human YTHDF proteins can functionally substitute for Pho92, but require multiple hydrophobic patches within the IDR for decay activity. Thus, on endogenous m6A-modified transcripts during meiosis, the promiscuous Pho92 IDR is restricted to a single functional interface, revealing unappreciated specificity in m6A-directed mRNA decay.

molecular biology↗

bioRxiv2025

A new double reporter strategy reveals a subset of non-migratory hematopoietic stem cells localized in a dynamic bone marrow niche.

Interactions of hematopoietic stem cells (HSCs) with the bone marrow microenvironment are critical to regulate stem cell function in homeostasis, emergency hematopoiesis and ageing. The dynamic behavior of endogenous HSCs in their niches has been challenging to study due to the complexity of markers required to define pure HSCs. Multiple recently developed reporter strategies advanced our capacity to identify HSCs in situ. Yet, they provide different levels of HSC enrichment and are frequently not stable in situations of perturbed hematopoiesis, leading to contradictory observations of HSC migratory behavior. Here we employed a new double reporter strategy by combining the Hoxb5-mKO2 and Vwf-GFP reporters, that are stably expressed in homeostasis, stress and ageing. Hoxb5+Vwf+ cells represent a subset of highly pure and potent long-term HSCs with a platelet-biased pattern of differentiation, that are included in the populations identified by other reporter strategies. Intravital microscopy revealed Hoxb5+Vwf+ cells to be non-migratory in homeostasis, following platelet depletion when these cells are actively proliferating, and during ageing. This non-migratory behavior of HSCs in homeostasis and stress indicates that their activation does not inherently depend on relocation to alternative niches eliciting proliferation, as previously proposed. We found Hoxb5+Vwf+ HSCs in direct contact with vasculature and LepR+ perivascular cells but not preferentially closer to megakaryocytes. Nevertheless, increased megakaryopoiesis following platelet depletion brings megakaryocytes into close proximity of Hoxb5+Vwf+ HSCs, revealing previously unrecognized niche dynamics in HSC regulation during regeneration. DATA SHARING STATEMENTOriginal data will be made available upon reasonable request. The RNA sequencing data used in this study has been published before35 and is available from GEO (GSE81682). The code used for analysis is available from https://github.com/loversaber/Secchi_Paper_RNAseq_Analysis.git. KEY POINTSO_LICombined expression of Hoxb5-mKO2 and Vwf-GFP reporters defines a population of highly enriched long-term HSCs in situ and in vivo. C_LIO_LIHoxb5+Vwf+ HSCs are non-migratory in homeostasis, stress hematopoiesis and ageing, while their niche is highly dynamic. C_LI

cell biology↗

bioRxiv2024

Impaired DNA damage response and inflammatory signalling underpins hematopoietic stem cell defects in Gata2 haploinsufficiency

Clinical GATA2 deficiency syndromes arise from germline haploinsufficiency inducing mutations in GATA2, resulting in immunodeficiency that evolves to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). How GATA2 haploinsufficiency disrupts the function and transcriptional network of hematopoietic stem/progenitors (HSCs/HSPCs) to facilitate the shift from immunodeficiency to pre-leukemia is poorly characterised. Using a conditional mouse model harboring a single allele deletion of Gata2 from the start of HSC development in utero, we identified pervasive defects in HSPC differentiation from young adult Gata2 haploinsufficient mice during B-cell development, early erythroid specification, megakaryocyte maturation to platelets and inflammatory cell generation. Gata2 haploinsufficiency abolished HSC self-renewal and multi-lineage differentiation capacity. These functional alterations closely associated with deregulated DNA damage responses and inflammatory signalling conveyed from Gata2 haploinsufficient HSCs. We identified genetic interplay between Gata2 and Asxl1, a driver of DNA damage and inflammation and, notably, a recurrent secondary mutation found in GATA2 haploinsufficiency disease progression to MDS/AML. shRNA mediated knockdown of Asxl1 in Gata2 haploinsufficient HSPCs led to an enhanced differentiation block in vitro. By analysis of HSCs from young adult compound Gata2/Asxl1 haploinsufficient mice, we discovered hyperproliferation of double haploinsufficient HSCs, which were also functionally compromised in transplantation compared to their single Gata2 or Asxl1 haploinsufficient counterparts. Through both Gata2/Asxl1 dependent and unique transcriptional programs, HSCs from compound Gata2/Asxl1 haploinsufficient fortified deregulated DNA damage responses and inflammatory signalling initiated in Gata2 haploinsufficient HSCs and established a broad pre-leukemic program. Our data reveal how Gata2 haploinsufficiency initially drives deregulation of HSC genome integrity and suggest the mechanisms of how secondary mutations like ASXL1 take advantage of HSC genomic instability to nurture a pre-leukemic state in GATA2 haploinsufficiency syndromes.

cancer biology↗

bioRxiv2022

Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

Current in vitro models of developmental blood formation lack spatio-temporal accuracy and weakly replicate successive waves of hematopoiesis. Herein, we describe a mouse embryonic stem cell (SC)-derived 3D hemogenic gastruloid (haemGx) that captures multi-wave blood formation, progenitor specification from hemogenic endothelium (HE), and generates hematopoietic progenitors capable of short-term engraftment of immunodeficient mice upon maturation in an in vivo niche. We took advantage of the haemGx model to interrogate the origins of infant acute myeloid leukemia (infAML). We focused on MNX1-driven leukemia, representing the commonest genetic abnormality unique to the infant group. Enforced MNX1 expression in haemGx promotes the expansion and in vitro transformation of yolk sac-like erythroid-myeloid progenitors at the HE-to-hematopoietic transition to faithfully recapitulate patient transcriptional signatures. By combining phenotypic, functional and transcriptional profiling, including at the single-cell level, we establish the haemGx as a useful new model for the study of normal and leukemic embryonic hematopoiesis.

developmental biology↗

bioRxiv2022

A time and single-cell resolved model of hematopoiesis

The paradigmatic tree model of hematopoiesis is increasingly recognized to be limited as it is based on heterogeneous populations and largely inferred from non-homeostatic cell fate assays. Here, we combine persistent labeling with time-series single-cell RNA-Seq to build the first real- time, quantitative model of in vivo tissue dynamics for any mammalian organ. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between single cell molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of neutrophil differentiation, illustrating how the new model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a Kinetoscope allows sequential images to merge into a movie. We posit that this approach is broadly applicable to empower single cell genomics to reveal important tissue scale dynamics information. HighlightsO_LICell flux analysis reveals high-resolution kinetics of native bone marrow hematopoiesis C_LIO_LIQuantitative model simulates cell behavior in real-time and connects it with gene expression patterns C_LIO_LIDistinct lineage-affiliated progenitors have unique self-renewal and differentiation properties C_LIO_LITransplanted HSCs display accelerated stage- and lineage-specific differentiation C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/506735v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@244ad6org.highwire.dtl.DTLVardef@ad632borg.highwire.dtl.DTLVardef@149daf9org.highwire.dtl.DTLVardef@1c7183f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Mannose metabolism inhibition sensitizes acute myeloid leukemia cells to cytarabine and FLT3 inhibitor therapy by modulating fatty acid metabolism to drive ferroptotic cell death.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a novel connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

cancer biology↗

bioRxiv2021

Peptidylarginine deiminase IV (PADI4) is not essential for cell-autonomous HSC maintenance and normal haematopoiesis

Peptidylarginine deiminases (PADIs, or PADs) are emerging as key regulators of human physiology and pathophysiology. The nuclear deiminase PADI4 regulates embryonic stem cell pluripotency, however its role in adult stem cells is unknown. PADI4 is expressed most highly in the bone marrow (BM), where it is found as part of a self-renewal-associated gene signature and shown to modulate the function of critical transcriptional regulators such as Tal1 and c-Myc, suggesting that it regulates haematopoietic development or regeneration. We investigated the functional significance of PADI4 in haematopoietic stem cell (HSC) biology and normal haematopoiesis. We employed two conditional mouse models of tissue-specific Padi4 ablation, where Padi4 was completely deleted either after the emergence of HSCs, or acutely in the BM of adult mice. We found that loss of PADI4 does not significantly affect HSC self-renewal or differentiation potential upon injury or serial transplantation, nor does it lead to exhaustion or premature ageing of HSCs. Thus, surprisingly, PADI4 is dispensable for cell-autonomous HSC maintenance, differentiation and haematopoietic regeneration. This work has important implications for the clinical use of PADI4 inhibitors as therapeutic agents in autoimmunity and cancer. Key PointsO_LIPADI4 is dispensable for steady-state and post-transplantation haematopoiesis C_LIO_LIHSCs do not require intrinsic PADI4 activity to respond to haematopoietic injury C_LIO_LIPADI4 deficiency does not lead to premature HSC ageing or exhaustion C_LI

developmental biology↗