Changes in the properties of membrane tethers in response to HP1α depletion in MCF7 cells.

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different.

Heterochromatin protein 1α interacts with parallel RNA and DNA G-quadruplexes.

The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4-DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome.

Depletion of HP1α alters the mechanical properties of MCF7 nuclei.

Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin.

Regulation of host-infection ability in the grass-symbiotic fungus Epichloë festucae by histone H3K9 and H3K36 methyltransferases.

Recent studies have identified key genes that control the symbiotic interaction between Epichloë festucae and Lolium perenne. Here we report on the identification of specific E. festucae genes that control host infection. Deletion of setB, which encodes a homologue of the H3K36 histone methyltransferase Set2/KMT3, reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homologue of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect L. perenne. Alleles that complement the culture and plant phenotypes of both mutants also complement the histone methylation defects. Co-inoculation of either ΔsetB or ΔclrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.

DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies.

Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3',5'-dinucleoside ß-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.

Regulation of subtelomeric fungal secondary metabolite genes by H3K4me3 regulators CclA and KdmB.

Studies on the regulation of fungal secondary metabolism highlight the importance of histone H3K4 methylation regulators Set1, CclA (Ash2) and KdmB (KDM5), but it remains unclear whether these proteins act by direct modulation of H3K4me3 at the target genes. In filamentous fungi, secondary metabolite genes are frequently located near telomeres, a site where H3K4 methylation is thought to have a repressive role. Here we analyzed the role of CclA, KdmB and H3K4me3 in regulating the subtelomeric EAS and LTM cluster genes in Epichloë festucae. Depletion of H3K4me3 correlated with transcriptional activation of these genes in ΔcclA, similarly enrichment of H3K4me3 correlated with transcriptional repression of the genes in ΔkdmB which was accompanied by significant reduction in the levels of the agriculturally undesirable lolitrems. These transcriptional changes could only be explained by the alterations in H3K4me3 and not in the subtelomerically-important marks H3K9me3/K27me3. However, H3K4me3 changes in both mutants were not confined to these regions but occurred genome-wide, and at other subtelomeric loci there were inconsistent correlations between H3K4me3 enrichment and gene repression. Our study suggests that CclA and KdmB are crucial regulators of secondary metabolite genes, but these proteins likely act via means independent to, or in conjunction with the H3K4me3 mark.

Heterochromatin protein 1 expression is reduced in human thyroid malignancy.

Owing to the loss of heterochromatin integrity that occurs during thyroid tumorigenesis, the expression of Heterochromatin Protein 1 isoforms HP1α and HP1ß was assessed by immunohistochemistry in 189 thyroid tumors and non-neoplastic tissues. Expression of HP1ß was significantly decreased in all thyroid lesions, except in follicular adenomas, when compared with matched adjacent normal tissue. This loss of HP1ß expression may in part be caused by microRNA dysregulation. An example is miR-205, a microRNA that is abundantly upregulated in thyroid carcinomas and shown to reduce the expression of HP1ß. In contrast to HP1ß, HP1α expression was only reduced in metastatic carcinomas and poorly differentiated lesions. These results suggest the reduction of HP1ß followed by a decrease in HP1α contributes to the pathogenesis of thyroid carcinomas, and their loss is a potential marker of thyroid malignancy and metastatic potential, respectively.

Inverted strand polarity yields thermodynamically stable G-quadruplexes and prevents duplex formation within extended DNA.

DNA G-quadruplexes (G4) formed in guanine-rich sequences play a key role in genome function and maintenance, interacting with multiple proteins. However, structural and functional studies of G4s within duplex DNA have been challenging because of the transient nature of G4s and thermodynamic preference of G-rich DNA to form duplexes with their complementary strand rather than G4s. To overcome these challenges, we have incorporated native nucleotides in G-rich sequences using commercially available inverted 3'-O-DMT-5'-O-phosphoramidites of native nucleosides, to give 3'-3' and 5'-5' linkages in the centre of the G-tract. Using circular dichroism and 1H nuclear magnetic resonance spectroscopies and native gel electrophoresis, we demonstrate that these polarity-inverted DNA sequences containing four telomeric repeats form G4s of parallel topology with one lateral or diagonal loop across the face of the quadruplex and two propeller loops across the edges of the quadruplex. These G4s were stable even in the presence of complementary C-rich DNA. As an example, G4 assemblies of inverted polarity were shown to bind to the hinge region of Heterochromatin Protein 1α (HP1α), a known G4-interacting domain. As such, internal polarity inversions in DNA provide a useful tool to control G4 topology while also disrupting the formation of other secondary structures, particularly the canonical duplex.

Ankyrin2 is essential for neuronal morphogenesis and long-term courtship memory in Drosophila.

Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

The normally antiviral enzyme APOBEC3A1-4 is an endogenous mutagen in many different human cancers5-7, where it becomes hijacked to fuel tumor evolvability. APOBEC3A's single-stranded DNA C-to-U editing activity1,8 results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations5-7. Transgenic expression in mice demonstrates its tumorigenic potential9. APOBEC3A inhibitors may therefore comprise a novel class of anti-cancer agents that work by blocking mutagenesis, preventing tumor evolvability, and lessening detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC recognition motif that is part of a three-nucleotide loop. The nuclease-resistant phosphorothioated derivatives of these inhibitors maintain nanomolar in vitro potency against APOBEC3A, localize to the cell nucleus, and block APOBEC3A activity in human cells. These results combine to suggest roles for these inhibitors to study A3A activity in living cells, potentially as conjuvants, leading toward next-generation, combinatorial anti-mutator and anti-cancer therapies.

The dynamic mobility of histone H1 is regulated by cyclin/CDK phosphorylation.

The linker histone H1 is involved in maintaining higher-order chromatin structures and displays dynamic nuclear mobility, which may be regulated by posttranslational modifications. To analyze the effect of H1 tail phosphorylation on the modulation of the histone's nuclear dynamics, we generated a mutant histone H1, referred to as M1-5, in which the five cyclin-dependent kinase phosphorylation consensus sites were mutated from serine or threonine residues into alanines. Cyclin E/CDK2 or cyclin A/CDK2 cannot phosphorylate the mutant in vitro. Using the technique of fluorescence recovery after photobleaching, we observed that the mobility of a green fluorescent protein (GFP)-M1-5 fusion protein is decreased compared to that of a GFP-wild-type H1 fusion protein. In addition, recovery of H1 correlated with CDK2 activity, as GFP-H1 mobility was decreased in cells with low CDK2 activity. Blocking the activity of CDK2 by p21 expression decreased the mobility of GFP-H1 but not that of GFP-M1-5. Finally, the level and rate of recovery of cyan fluorescent protein (CFP)-M1-5 were lower than those of CFP-H1 specifically in heterochromatic regions. These data suggest that CDK2 phosphorylates histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions.

PPM1B depletion induces premature senescence in human IMR-90 fibroblasts.

p53 and NF-κB are key transcription factors in regulating the gene expression program of cellular and organismal senescence. PPM1B is a member of the protein phosphatase 2C family and plays a role in negatively regulating p53 and NF-κB thereby possibly attenuating the gene expression program of cellular senescence. Here, possible involvement of PPM1B in replicative senescence has been investigated using the in vitro aging model of IMR-90 cells. PPM1B protein levels are progressively decreased in a replicative age-dependent manner. Importantly, PPM1B depletion induces a robust senescence phenotype as evidenced by significant growth arrest and senescence marker expression. Given that PPM1B depletion-induced senescence is partially rescued by inactivating p38 MAPK, our results identify PPM1B as a critical regulator of both p38 MAPK-dependent and independent senescence pathways during normal cellular aging process.

Helicases, G4-DNAs, and drug design.

New helicase assays that recognise therapeutically important G4-DNA structures will lead to the discovery of novel molecular entities that bind not only to G4-tetrads, but also to grooves and loops of G4-DNA. Such assays can also provide inhibitors of G4-specific helicases that will shed light on the emerging involvement of helicases in cancer and other diseases linked to defective DNA repair pathways.

Assembly Dependent Fluorescence Enhancing Nucleic Acids in Sequence-Specific Detection of Double-Stranded DNA.

In this study the position of the thiazole orange derivative in triplex-forming oligonucleotides (TFOs) is varied and the fluorescence of the resulting complexes with DNA duplexes, single-stranded DNAs and RNAs are evaluated. Under similar conditions single attachment of the TO-dye to 2'-O-propargyl nucleotides in the TFOs (assembly dependent fluorescence enhancing nucleic acids, AFENA) led to probes with low fluorescent intensity in the single-stranded state with fluorescence quantum yield (ΦF ) of 0.9 %-1.5 %. Significant increase in fluorescence intensity was detected after formation of DNA triplexes (ΦF =23.5 %-34.9 %). Under similar conditions, Watson-Crick-type duplexes formed by the probes with single stranded (ss) RNA and ssDNA showed lower fluorescence intensities. Bugle insertions of twisted intercalating nucleic acid (TINA) monomers were shown to improve the fluorescent characteristics of GT/GA-containing antiparallel AFENA-TFOs. Self-aggregation of TFOs caused by guanosines was eliminated by TINA insertion which also promoted DNA triplex formation at pH 7.2. Importantly these AFENA-TINA-TFOs can bind to the duplex in the presence of complementary RNA at 37 °C.

Assembly Dependent Fluorescence Enhancing Nucleic Acids in Sequence-Specific Detection of Double-Stranded DNA.

Invited for this month's cover is the group of Dr. Vyacheslav V. Filichev from Massey University, New Zealand and a collaborator from the Instituto de Química Física Rocasalano, CSIC, Spain. The cover picture shows how a DNA strand that forms a highly stable G-quadruplex can be converted into an efficient DNA triplex-forming oligonucleotide by incorporating a pyrene intercalator into the sequence. Read the full text of the article at 10.1002/cplu.201300310.

Histone H1 subtypes differentially modulate chromatin condensation without preventing ATP-dependent remodeling by SWI/SNF or NURF.

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.

Phosphorylation of the linker histone H1 by CDK regulates its binding to HP1alpha.

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.