Biology subjects

Krupalnik, V.

Publications and source records attributed to Krupalnik, V..

5 recordsLinked to original sources

bioRxiv2020

Context-dependent functional compensation between Ythdf m6A readers

The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay, translation and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is dynamically regulated by a set of writer, eraser and reader proteins. The YTH-domain family of proteins: Ythdf1, Ythdf2, and Ythdf3, are three homologous m6A binding proteins, which have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer for each of the Ythdf readers and for the three readers together (triple-KO). We then estimated the effect in-vivo, in mouse gametogenesis and viability, and in-vitro, in mouse embryonic stem cells (mESCs). We show that in gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, possibly due to differences in readers expression, both in quantity and in spatial location. By knocking out the three readers together and systematically testing offspring genotypes, we have revealed a redundancy in the readers role during early development, a redundancy which is dosage-dependent. Additionally, we show that in mESCs there is compensation between the three readers, since the inability to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a novel model for the Ythdf readers function. There is a dosage-dependent redundancy when all three readers are co-expressed in the same location in the cells.

developmental biology↗

bioRxiv2020

Tripartite Inhibition of SRC-WNT-PKC Signalling Consolidates Human Naive Pluripotency

Different conditions have been devised to isolate MEK/ERK signalling independent human naive pluripotent stem cells (PSCs) that are distinct from conventional primed PSCs and better correspond to pre-implantation developmental stages. While the naive conditions described thus far endow human PSCs with different extents of naivety features, isolating human pluripotent cells that retain characteristics of ground state pluripotency while maintaining differentiation potential and genetic integrity, remains a major challenge. Here we engineer reporter systems that allow functional screening for conditions that can endow both the molecular and functional features expected from human naive pluripotency. We establish that simultaneous inhibition of SRC-NF{kappa}B, WNT/{beta}CATENIN and PKC signalling pathways is essential for enabling expansion of teratoma competent fully naive human PSCs in defined or xeno-free conditions. Divergent signalling and transcriptional requirements for maintaining naive pluripotency were found between mouse and human. Finally, we establish alternative naive conditions in which MEK/ERK inhibition is substituted with inhibition for NOTCH/RBPj signalling, which allow obtaining alternative human naive PSCs with diminished risk for loss of imprinting and deleterious global DNA hypomethylation. Our findings set a framework for the signalling foundations of human naive pluripotency and may advance its utilization in future translational applications. Highlights of key findingsO_LICombined inhibition of SRC, WNT and PKC signaling consolidates human naive pluripotency C_LIO_LIStable expansion of DNA/RNA methylation-independent and TGF/ACTIVIN-independent human naive PSCs C_LIO_LIOpposing roles for ACTIVIN and WNT/{beta}CATENIN signaling on mouse vs. human naive pluripotency C_LIO_LI2i and MEK/ERKi independent alternative human naive PSC conditions via inhibiting NOTCH/RBPj signaling C_LI

developmental biology↗

bioRxiv2018

Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency

The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive complex involved in many pathological and physiological processes in the cell. Previous studies have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein followed by applying naive ground-state pluripotency conditions, results in a highly efficient and near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to block Mbd3-dependent NuRD activity by identifying additional functionally relevant components of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also known as P66), a relatively uncharacterized NuRD-specific subunit, whose complete deletion does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency. Complete ablation of Gatad2a in somatic cells, but not Gatad2b, results in a deterministic naive iPSC reprogramming where up to 100% of donor somatic cells successfully complete the process within 8 days. Genetic and biochemical analysis established a distinct sub-complex within the NuRD complex (Gatad2a-Chd4-Mbd3) as the functional and biochemical axis blocking reestablishment of murine naive pluripotency. Disassembly of this axis by depletion of Gatad2a, results in resistance to conditions promoting exit of naive pluripotency and delays differentiation. We further highlight context- and posttranslational dependent modifications of the NuRD complex affecting its interactions and assembly in different cell states. Collectively, our work unveils the distinct functionality, composition and interactions of Gatad2a-Chd4-Mbd3/NuRD subcomplex during the resolution and establishment of mouse naive pluripotency.

developmental biology↗

bioRxiv2017

High-Resolution Dissection of Conducive Reprogramming Trajectory to Ground State Pluripotency

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) with four transcription factors Oct4, Sox2, Klf4 and cMyc (abbreviated as OSKM)1 has provoked interest to define the molecular characteristics of this process2-7. Despite important progress, the dynamics of epigenetic reprogramming at high resolution in correctly reprogrammed iPSCs and throughout the entire process remain largely undefined. This gap in understanding results from the inefficiency of conventional reprogramming methods coupled with the difficulty of prospectively isolating the rare cells that eventually correctly reprogram into iPSCs. Here we characterize cell fate conversion from fibroblast to iPSC using a highly efficient deterministic murine reprogramming system engineered through optimized inhibition of Gatad2a-Mbd3/NuRD repressive sub-complex. This comprehensive characterization provides single-day resolution of dynamic changes in levels of gene expression, chromatin modifications, TF binding, DNA accessibility and DNA methylation. The integrative analysis identified two transcriptional modules that dominate successful reprogramming. One consists of genes whose transcription is regulated by on/off epigenetic switching of modifications in their promoters (abbreviated as ESPGs), and the second consists of genes with promoters in a constitutively active chromatin state, but a dynamic expression pattern (abbreviated as CAPGs). ESPGs are mainly regulated by OSK, rather than Myc, and are enriched for cell fate determinants and pluripotency factors. CAPGs are predominantly regulated by Myc, and are enriched for cell biosynthetic regulatory functions. We used the ESPG module to study the identity and temporal occurrence of activating and repressing epigenetic switching during reprogramming. Removal of repressive chromatin modifications precedes chromatin opening and binding of RNA polymerase II at enhancers and promoters, and the opposite dynamics occur during repression of enhancers and promoters. Genome wide DNA methylation analysis demonstrated that de novo DNA methylation is not required for highly efficient conducive iPSC reprogramming, and identified a group of super-enhancers targeted by OSK, whose early demethylation marks commitment to a successful reprogramming trajectory also in inefficient conventional reprogramming systems. CAPGs are distinctively regulated by multiple synergystic ways: 1) Myc activity, delivered either endogenously or exogenously, dominates CAPG expression changes and is indispensable for induction of pluripotency in somatic cells; 2) A change in tRNA codon usage which is specific to CAPGs, but not ESPGs, and favors their translation. In summary, our unbiased high-resolution mapping of epigenetic changes on somatic cells that are committed to undergo successful reprogramming reveals interleaved epigenetic and biosynthetic reconfigurations that rapidly commission and propel conducive reprogramming toward naive pluripotency.

developmental biology↗

bioRxiv2017

OCT4 Impedes Cell Fate Redirection By The Melanocyte Lineage Master Regulator MITF

Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has never been systemically examined. Here we discovered that amongst different developmental stages, embryonic stem cells (ESCs) were resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. We generated a transgenic system and found that in ESCs, the pluripotency master regulator, OCT4, counteracts pro-differentiation induced by MITF by physical interference with MITF transcriptional activity. We further found that ESCs must be released from OCT4-maintained pluripotency prior to ectopically induced differentiation. Moreover, OCT4 induction in various differentiated cells repressed their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggested that OCT4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.

developmental biology↗