HA95 and LAP2β mediate a novel chromatin–nuclear envelope interaction implicated in initiation of DNA replication

HA95 is a chromatin-associated protein that interfaces the nuclear envelope (NE) and chromatin. We report an interaction between HA95 and the inner nuclear membrane protein lamina-associated polypeptide (LAP) 2β, and a role of this association in initiation of DNA replication. Precipitation of GST–LAP2β fusion proteins and overlays of immobilized HA95 indicate that a first HA95-binding region lies within amino acids 137–242 of LAP2β. A second domain sufficient to bind HA95 colocalizes with the lamin B–binding domain of LAP2β at residues 299–373. HA95–LAP2β interaction is not required for NE formation. However, disruption of the association of HA95 with the NH2-terminal HA95-binding domain of LAP2β abolishes the initiation, but not elongation, of DNA replication in purified G1 phase nuclei incubated in S-phase extract. Inhibition of replication initiation correlates with proteasome-mediated proteolysis of Cdc6, a component of the prereplication complex. Rescue of Cdc6 degradation with proteasome inhibitors restores replication. We propose that an interaction of LAP2β, or LAP2 proteins, with HA95 is involved in the control of initiation of DNA replication

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2′-modified-4′-thionucleosides

AbstractGene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2′-modified-4′-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2′-fluoro-4′-thionucleoside and 2′-fluoronucleoside units as well as only 2′-fluoronucleoside units, while those prepared as combination of 2′-OMe nucleoside/2′-OMe-4′-thionucleoside and 2′-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen–induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokine signaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.Yoshimoto T., Chaya T., Varner L.R., et al. The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina. Journal of Biological Chemistry 299, 105461 (2023); https://doi.org/10.1016/j.jbc.2023.105461

Rad9 modulates the P21WAF1 pathway by direct association with p53

<p>Abstract</p> <p>Background</p> <p>Previous studies suggest that human <it>RAD9 </it>(hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival <it>P21</it>^{<it>WAF1</it>}. This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of <it>P21</it>^{<it>WAF1</it>}.</p> <p>Results</p> <p>The transfection of phosphorylation-defective <it>hRAD9 </it>mutants of C-terminus resulted in reduction of the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transactivation; the knockdown of total hRad9 elicited an increased <it>P21</it>^{<it>WAF1 </it>}mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5' region of <it>P21</it>^{<it>WAF1 </it>}gene. The association was reduced in the experiment of phosphorylation-defective <it>hRAD9 </it>mutants.</p> <p>Conclusion</p> <p>The present study indicates the direct involvement of hRad9 in the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.</p

Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionucleosides

Gene suppression via U1 small nuclear RNA interference (U1i) is considered to be one of the most attractive approaches, and takes the place of general antisense, RNA interference (RNAi), and anti-micro RNA machineries. Since the U1i can be induced by short oligonucleotides (ONs), namely U1 adaptors consisting of a ‘target domain’ and a ‘U1 domain’, we prepared adaptor ONs using 2'-modified-4'-thionucleosides developed by our group, and evaluated their U1i activity. As a result, the desired gene suppression via U1i was observed in ONs prepared as a combination of 2'-fluoro-4'-thionucleoside and 2'-fluoronucleoside units as well as only 2'-fluoronucleoside units, while those prepared as combination of 2'-OMe nucleoside/2'-OMe-4'-thionucleoside and 2'-fluoronucleoside units did not show significant activity. Measurement of Tm values indicated that a higher hybridization ability of adaptor ONs with complementary RNA is one of the important factors to show potent U1i activity

Skein Theory and Topological Quantum Registers: Braiding Matrices and Topological Entanglement Entropy of Non-Abelian Quantum Hall States

We study topological properties of quasi-particle states in the non-Abelian quantum Hall states. We apply a skein-theoretic method to the Read--Rezayi state whose effective theory is the SU(2)_K Chern--Simons theory. As a generalization of the Pfaffian (K=2) and the Fibonacci (K=3) anyon states, we compute the braiding matrices of quasi-particle states with arbitrary spins. Furthermore we propose a method to compute the entanglement entropy skein-theoretically. We find that the entanglement entropy has a nontrivial contribution called the topological entanglement entropy which depends on the quantum dimension of non-Abelian quasi-particle intertwining two subsystems.Comment: 42 pages, many eps file

Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4′-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4′-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4′-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response