Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR–DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (γH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.     

Chlorophyll biosynthesis. Expression of a second chl I gene of magnesium chelatase in Arabidopsis supports only limited chlorophyll synthesis

Magnesium (Mg) chelatase is a heterotrimeric enzyme complex that catalyzes a key regulatory and enzymatic reaction in chlorophyll biosynthesis, the insertion of Mg(2+) into protoporphyrin IX. Studies of the enzyme complex reconstituted in vitro have shown that all three of its subunits, CHL I, CHL D, and CHL H, are required for enzymatic activity. However, a new T-DNA knockout mutant of the chlorina locus, ch42-3 (Chl I), in Arabidopsis is still able to accumulate some chlorophyll despite the absence of Chl I mRNA and protein. In barley (Hordeum vulgare), CHL I is encoded by a single gene. We have identified an open reading frame that apparently encodes a second Chl I gene, Chl I2. Chl I1 and Chl I2 mRNA accumulate to similar levels in wild type, yet CHL I2 protein is not detectable in wild type or ch42-3, although the protein is translated and stromally processed as shown by in vivo pulse labeling and in vitro chloroplast imports. It is surprising that CHL D accumulates to wild-type levels in ch42-3, which is in contrast to reports that CHL D is unstable in CHL I-deficient backgrounds of barley. Our results show that limited Mg chelatase activity and CHL D accumulation can occur without detectable CHL I, despite its obligate requirement in vitro and its proposed chaperone-like stabilization and activation of CHL D. Thus, the unusual post-translational regulation of the CHL I2 protein provides an opportunity to study the different steps involved in stabilization and activation of the heterotrimeric Mg chelatase in vivo.     

Chlornaphazine and chlorambucil induce dominant lethal mutations in male mice

Two antineoplastic agents, chlornaphazine (CN) and chlorambucil (CHL), were tested for the induction of dominant lethal mutations in male mice. Both compounds are nitrogen mustard derivatives and have been shown to be genotoxic in a variety of organisms. CN was administered intraperitoneally to DBA/2J male mice at a dosage of 0, 500, 1000, or 1500 mg/kg body weight (bw). Immediately following treatment, each male was mated at 4-day intervals to two virgin C57BL/6J females. CHL was administered intraperitoneally to C3H/HeJ and DBA/2J males at a dosage of 0, 2.5, or 5.0 mg/kg bw. These males were mated at weekly intervals to two virgin T-stock females. CN and CHL clearly induced dominant lethal mutations. CN induced dominant lethal effects in all post-meiotic germ-cell stages of treated DBA males, with a clear dose-response relationship. The results with CHL-treated DBA males indicated that all post-meiotic germ-cell stages, except late-spermatids, were affected by CHL treatment, while in C3H males, CHL induced dominant lethal effects in all post-meiotic germ-cell stages. A dose-response relationship was also observed with CHL in C3H male mice. In the present experiments, regardless of the agent or the mouse strain used, spermatids appeared to be the germ-cell stage most sensitive to dominant lethal induction.     

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Deltaynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 muM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K (m) for nitrate of about 125 muM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 muM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Deltaynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Deltaynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.     

VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage

All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM.     

Neural recognition molecules CHL1 and NB-3 regulate apical dendrite orientation in the neocortex via PTP alpha

Apical dendrites of pyramidal neurons in the neocortex have a stereotypic orientation that is important for neuronal function. Neural recognition molecule Close Homolog of L1 (CHL1) has been shown to regulate oriented growth of apical dendrites in the mouse caudal cortex. Here we show that CHL1 directly associates with NB-3, a member of the F3/contactin family of neural recognition molecules, and enhances its cell surface expression. Similar to CHL1, NB-3 exhibits high-caudal to low-rostral expression in the deep layer neurons of the neocortex. NB-3-deficient mice show abnormal apical dendrite projections of deep layer pyramidal neurons in the visual cortex. Both CHL1 and NB-3 interact with protein tyrosine phosphatase alpha (PTPalpha) and regulate its activity. Moreover, deep layer pyramidal neurons of PTPalpha-deficient mice develop misoriented, even inverted, apical dendrites. We propose a signaling complex in which PTPalpha mediates CHL1 and NB-3-regulated apical dendrite projection in the developing caudal cortex.     

Interaction of amphiphilic chlorin-based photosensitizers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine monolayers

The drawbacks of the presently used photosensitizers include their relatively low selectivity toward cancer cells, and long-lasting accumulation in healthy tissues. Our recent results indicate that conjugating a photosensitizer with folic acid both enhances the active uptake by cells, and decreases the accumulation in healthy tissue. Here, the interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers used as model membranes, and three different photosensitizers were studied; the derivatives were the non-conjugated meta-tetrahydroxyphenylchlorin (m-THPC, CHL1) and tris(3-hydroxyphenyl)-4-carboxyphenylchlorin (CHL2), as well as a folic acid-conjugated m-THPC-like molecule (CHL3). The results obtained indicate that the folate moiety present in the conjugated derivative CHL3 is involved in the interaction with the phospholipid polar heads. This interaction may be responsible for a better miscibility of CHL3 with the DPPC films compared to CHL1 and CHL2, while elimination of CHL3 from the tissue may be due rather to specific, biological processes and not to its polarity.     

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.     

Euchromatic subdomains in rice centromeres are associated with genes and transcription

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.     

Two distinct methods analyzing chromatin structure using centrifugation and antibodies to modified histone H3: both provide similar chromatin states of the Rit1/Bcl11b gene

Chromatin state of a 2-Mb region harboring Rit1/Bcl11b on mouse chromosome 12 was examined using two distinct methods. One is ChIP assay examining the degree of enrichment with histone H3 methylated at lysine 9 (H3-mLys9) in chromatin and the other is H/E (heterochromatin/euchromatin) assay that measures a chromatin condensation state by using centrifugation. The ChIP assay showed that a 50-kb interval covering the gene and an upstream region constituted chromatin enriched with unmethylated H3-mLys9 in cells expressing Rit1 compared to cells not expressing Rit1. In contrast, regions other than the 50-kb interval did not show much difference in the enrichment between the two different types of cells. On the other hand, H/E assay of two expressing and two non-expressing tissues provided compatible fractionation patterns, suggesting that the chromatin condensation state detected by H/E assay is correlated with the chromatin state controlled by histone H3 tail modification linked to gene expression. These results indicate that the centrifugation-based H/E assay should provide a new approach to the regulation of chromatin structure with respect to its condensation state, complementing ChIP assays.     

Computational site‐directed mutagenesis studies of the role of the hydrophobic triad on substrate binding in cholesterol oxidase

Cholesterol oxidase (ChOx) is a flavoenzyme that oxidizes and isomerizes cholesterol (CHL) to form cholest‐4‐en‐3‐one. Molecular docking and molecular dynamics simulations were conducted to predict the binding interactions of CHL in the active site. Several key interactions (E361‐CHL, N485‐FAD, and H447‐CHL) were identified and which are likely to determine the correct positioning of CHL relative to flavin‐adenine dinucleotide (FAD). Binding of CHL also induced changes in key residues of the active site leading to the closure of the oxygen channel. A group of residues, Y107, F444, and Y446, known as the hydrophobic triad, are believed to affect the binding of CHL in the active site. Computational site‐directed mutagenesis of these residues revealed that their mutation affects the conformations of key residues in the active site, leading to non‐optimal binding of CHL and to changes in the structure of the oxygen channel, all of which are likely to reduce the catalytic efficiency of ChOx. Proteins 2017; 85:1645–1655. © 2017 Wiley Periodicals, Inc.