Characterisation of KDM4A/JMJD2A as a histone demethylase in D.melanogaster

Histone sind basische Proteine, die im Zellkern von Eukaryoten vorkommen. Sie sind als Bestandteil des Chromatins für die Verpackung der DNA (es sind Spulen, um welche sich die DNA windet). Die Oktamer Einheit der Core-Partikel besteht aus zwei Kopien von jedem der vier Histone H2A, H2B, H3 und H4. Das fünfte Histon, welches H1 oder Linker Histon genannt wird, ist ein sehr Lysin-reiches Protein, welches mit der DNA-Aufnahme und der Beendigung der Nukleosomen Core-Partikel interagiert. Das H1 Histon hat Aufgaben in vielen verschiedenen Prozessen in unterschiedlichen Organismen. Es gibt viele Beweise, dass die Histone H1 zu dem so genannten Histon-Code beitragen. H1 Histone sind, ebenso wie die Core-Histone, Ziele von mehreren posttranslationalen Modifikationen (PTM), einschließlich der Phosphorylierung, Lysin-Methylierung und ADP-Ribosylierung. Kürzlich entdeckte, Trojer und Kollegen zeigten, dass Mitglieder der Unterfamilie der JMJD2/KDM4( lysin demethylase 4) jumonji-C-Typ Histon demethlylases trimethylated H1.4K26 der Säugetiere zu Di-und monomethylierte Staaten reduziert werden. Sie zeigten zum ersten Mal, dass H1 in Säugetieren demetyhlated werden kann. Bis heute gibt es keinen Bericht, der Demethylierung von Histon H1 in D. melanogaster beschreibt. In meinem Projekt untersuchten wir mögliche Demethylierung Aktivität von Lysin an Position 27) an Histon H1 und wir benutzten Drosophila melanogaster als Modellorganismus. In meinem Projekt wollten wir untersuchen, ob eine Demethylierung Aktivität von Säugetieren JMJD2A Homolog Targeting H1K27me2 auch in Drosophila zu erkennen ist.In eukaryotic cells, DNA is a part of a nuclear structure called chromatin. Chromatin was found to contain both nucleic acids and a series of acid soluble proteins that were termed “histones”. The histone octamer moiety of the core particle consists of two copies of each of the four histones H2A, H2b, H3 and H4. The fifth histone, termed H1 or linker histone,very lysine-rich protein, interacts with DNA entering and exiting the nucleosomal core particle. Histone linker H1 has roles in many different processes in different organisms, such as gene expression regulation in mammals, position effect variegation in mouse, etc. There are many evidences that also H1 histones contribute to the, so called, Histone code, combinatorial nature of histone amino- terminal modifications that many considerably extend the information potential of the genetic (DNA) code, and just as in the case of core histones further studies are necessary to decipher this code. H1 histones are, just as the core histones, targets of several posttranslational modifications (PTMs), including phosphorylation, lysine methylation and ADP-ribosylation. Recently, Trojer and collegues discovered and showed that members of the JMJD2/KDM4 (lysine demethylase 4) subfamily of jumonji-C type histone demethlylases reduce mammalian trimethylated H1.4K26 to di- and monomethylated states. They showed for the first time that H1 can be demetyhlated in mammals. To date there is no report describing demethylation of histone H1 in D. melanogaster. In my project we investigated potential demethylation activity of lysine at position 27) of linker histone H1 and we used Drosophila melanogaster as a model organism. we Wanted to investigate if there is a demethylation activity of mammalian JMJD2A homologue targeting H1K27me2 also in Drosophila

Phosphatidylinositol-4,5-Biphosphate-Dependent Rearrangement of TRPV4 Cytosolic Tails Enables Channel Activation by Physiological Stimuli

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP$_2$), although the structural rearrangements occurring on PIP$_2$ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP$_2$ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence $^{121}$KRWRK$^{125}$, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP$_2$ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP$_2$ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP$_2$-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP$_2$ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP$_2$ interacting site and conditions that deplete PIP$_2$ or restrict access of TRPV4 to PIP$_2$—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.Molecular and Cellular Biolog

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

International audienceActin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensi-tive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca 2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2. mechanotransduction | calcium signaling | RhoA | actin stress fibers | cance

Piezo2 channel regulates RhoA and actin cytoskeleton to promote cell mechanobiological responses

Actin polymerization and assembly into stress fibers (SFs) is central to many cellular processes. However, how SFs form in response to the mechanical interaction of cells with their environment is not fully understood. Here we have identified Piezo2 mechanosensitive cationic channel as a transducer of environmental physical cues into mechanobiological responses. Piezo2 is needed by brain metastatic cells from breast cancer (MDA-MB-231-BrM2) to probe their physical environment as they anchor and pull on their surroundings or when confronted with confined migration through narrow pores. Piezo2-mediated Ca2+ influx activates RhoA to control the formation and orientation of SFs and focal adhesions (FAs). A possible mechanism for the Piezo2-mediated activation of RhoA involves the recruitment of the Fyn kinase to the cell leading edge as well as calpain activation. Knockdown of Piezo2 in BrM2 cells alters SFs, FAs, and nuclear translocation of YAP; a phenotype rescued by overexpression of dominant-positive RhoA or its downstream effector, mDia1. Consequently, hallmarks of cancer invasion and metastasis related to RhoA, actin cytoskeleton, and/or force transmission, such as migration, extracellular matrix degradation, and Serpin B2 secretion, were reduced in cells lacking Piezo2

Mechanotransduction pulls the strings of matrix degradation at invadosome

International audienc

Mechanotransduction pulls the strings of matrix degradation at invadosome

International audienc