Chromatin transitions triggered by LH density as epigenetic regulators of the genome

Motivated by experiments connecting linker histone (LH) deficiency to lymphoma progression and retinal disorders, we study by mesoscale chromatin modeling how LH density (ρ) induces gradual, as well sudden, changes in chromatin architecture and how the process depends on DNA linker length, LH binding dynamics and binding mode, salt concentration, tail modifications, and combinations of ρ and linker DNA length. We show that ρ tightly regulates the overall shape and compaction of the fiber, triggering a transition from an irregular disordered state to a compact and ordered structure. Such a structural transition, resembling B to A compartment transition connected with lymphoma of B cells, appears to occur around ρ = 0.5. The associated mechanism is DNA stem formation by LH binding, which is optimal when the lengths of the DNA linker and LH C-terminal domain are similar. Chromatin internal and external parameters are key regulators, promoting or impeding the transition. The LH density thus emerges as a critical tunable variable in controlling cellular functions through structural transitions of the genome.     

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.     

Discerning non-disjunction in Down syndrome patients by means of GluK1-(AGAT)n and D21S2055-(GATA)n microsatellites on chromosome 21

INTRODUCTION:

Down syndrome (DS), the leading genetic cause of mental retardation, stems from non-disjunction of chromosome 21.

AIM:

Our aim was to discern non-disjunction in DS patients by genotyping GluK1-(AGAT)_n and D21S2055-(GATA)_n microsatellites on chromosome 21 using a family-based study design.

MATERIALS AND METHODS:

We have used a PCR and automated DNA sequencing followed by appropriate statistical analysis of genotype data for the present study

RESULTS AND DISCUSSION:

We show that a high power of discrimination and a low probability of matching indicate that both markers may be used to distinguish between two unrelated individuals. That the D21S2055-(GATA)_n allele distribution is evenly balanced, is indicated by a high power of exclusion [PE=0.280]. The estimated values of observed heterozygosity and polymorphism information content reveal that relative to GluK1-(AGAT)_n[H_obs=0.286], the D21S2055- (GATA)_n[H_obs=0.791] marker, is more informative. Though allele frequencies for both polymorphisms do not conform to Hardy-Weinberg equilibrium proportions, we were able to discern the parental origin of non-disjunction and also garnered evidence for triallelic (1:1:1) inheritance. The estimated proportion of meiosis-I to meiosis-II errors is 2:1 in maternal and 4:1 in paternal cases for GluK1-(AGAT)_n, whereas for D21S2055-(GATA)_n, the ratio is 2:1 in both maternal and paternal cases. Results underscore a need to systematically evaluate additional chromosome 21-specific markers in the context of non-disjunction DS.     

Induction of primary human T cell responses against hepatitis C virus-derived antigens NS3 or core by autologous dendritic cells expressing hepatitis C virus antigens: potential for vaccine and immunotherapy

Hepatitis C virus (HCV)-specific T cell responses have been suggested to play significant role in viral clearance. Dendritic cells (DCs) are professional APCs that play a major role in priming, initiating, and sustaining strong T cell responses against pathogen-derived Ags. DCs also have inherent capabilities of priming naive T cells against given Ags. Recombinant adenoviral vectors containing HCV-derived Core and NS3 genes were used to endogenously express HCV Core and NS3 proteins in human DCs. These HCV Ags expressing DCs were used to prime and stimulate autologous T cells obtained from uninfected healthy donors. The DCs expressing HCV Core or NS3 Ags were able to stimulate T cells to produce various cytokines and proliferate in HCV Ag-dependent manner. Evidence of both CD4(+) and CD8(+) T cell responses against HCV Core and NS3 generated in vitro were obtained by flow cytometry and Ab blocking experiments. Further, in secondary assays, the T cells primed in vitro exhibited HCV Ag-specific proliferative responses against recombinant protein Ags and also against immunodominant permissive peptide epitopes from HCV Ags. In summary, we demonstrate that the dendritic cells expressing HCV Ags are able to prime the Ag-specific T cells from uninfected healthy individuals in vitro. These studies have implications in designing cellular vaccines, T cell adoptive transfer therapy or vaccine candidates for HCV infection in both prophylactic and therapeutic settings.     

A multifunctional lentiviral-based gene knockdown with concurrent rescue that controls for off-target effects of RNAi

The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 (α-ACTN1) expression in human T cells. α-ACTN1 RNAi resulted in inhibited chemotaxis to SDF-1α, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTN1 (rr-α-ACTN1) in the same cell. The presence of a GFP tag on rr-α-ACTN1 allowed for detection of appropriate subcellular localization of rr-α-ACTN1. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.     

Biodegradation of chloromethane by Pseudomonas aeruginosa strain NB1 under nitrate-reducing and aerobic conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (+/-0.06) (mean +/- standard deviation) and 0.28 (+/-0.01) mg of total suspended solids (TSS) mg of CM(-1) under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (+/-0.10) electron equivalents (eeq) of NO(3)(-) per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 +/- 0.05 mol of N(2) per mol of NO(3)(-)). The stoichiometry of oxygen use with CM (0.85 +/- 0.21 eeq of O(2) per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (+/-0.06) micromol of CM mg of TSS(-1) day(-1), the maximum specific growth rate (micro(max)) was 0.0506 day(-1), and the Monod half-saturation coefficient (K(s)) was 0.067 (+/-0.004) microM. Under aerobic conditions, the values for k, micro(max), and K(s) were 10.7 (+/-0.11) micromol of CM mg of TSS(-1) day(-1), 0.145 day(-1), and 0.93 (+/-0.042) microM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Isolation and molecular detection of methylotrophic bacteria occurring in the human mouth

Diverse methylotrophic bacteria were isolated from the tongue, and supra- and subgingival plaque in the mouths of volunteers and patients with periodontitis. One-carbon compounds such as dimethylsulfide in the mouth are likely to be used as growth substrates for these organisms. Methylotrophic strains of Bacillus, Brevibacterium casei, Hyphomicrobium sulfonivorans, Methylobacterium, Micrococcus luteus and Variovorax paradoxus were characterized physiologically and by their 16S rRNA gene sequences. The type strain of B. casei was shown to be methylotrophic. Enzymes of methylotrophic metabolism were characterized in some strains, and activities consistent with growth using known pathways of C1-compound metabolism demonstrated. Genomic DNA from 18 tongue and dental plaque samples from nine volunteers was amplified by the polymerase chain reaction using primers for the 16S rRNA gene of Methylobacterium and the mxaF gene of methanol dehydrogenase. MxaF was detected in all nine volunteers, and Methylobacterium was detected in seven. Methylotrophic activity is thus a feature of the oral bacterial community.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype.