Juntao Gao

Allelic reprogramming of 3D chromatin architecture during early mammalian development

In mammals, chromatin organization undergoes drastic reprogramming after fertilization. However, the three-dimensional structure of chromatin and its reprogramming in preimplantation development remain poorly understood. Here, by developing a low-input Hi-C (genome-wide chromosome conformation capture) approach, we examined the reprogramming of chromatin organization during early development in mice. We found that oocytes in metaphase II show homogeneous chromatin folding that lacks detectable topologically associating domains (TADs) and chromatin compartments. Strikingly, chromatin shows greatly diminished higher-order structure after fertilization. Unexpectedly, the subsequent establishment of chromatin organization is a prolonged process that extends through preimplantation development, as characterized by slow consolidation of TADs and segregation of chromatin compartments. The two sets of parental chromosomes are spatially separated from each other and display distinct compartmentalization in zygotes. Such allele separation and allelic compartmentalization can be found as late as the 8-cell stage. Finally, we show that chromatin compaction in preimplantation embryos can partially proceed in the absence of zygotic transcription and is a multi-level hierarchical process. Taken together, our data suggest that chromatin may exist in a markedly relaxed state after fertilization, followed by progressive maturation of higher-order chromatin architecture during early development.

Kinetochore-Independent Chromosome Poleward Movement during Anaphase of Meiosis II in Mouse Eggs

Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase.

Pycnogenol® in cigarette filters scavenges free radicals and reduces mutagenicity and toxicity of tobacco smoke in vivo:

Despite large-scale anti-smoking campaigns throughout the world, the number of smokers remains high and cigarette smoking continues to represent a life-threatening health risk. Until a smoke-free society is achieved, reduction of cigarette smoke toxins may reduce the health burden. Current cigarette filter techniques are limited to the reduction of volatile tar constituents by dilution and by condensation on the filter surface. Vast quantities of harmful constituents, such as polycyclic aromatic hydrocarbons, heterocyclic (aromatic) amines, free radicals and reactive oxygen species, are inefficiently retained in the filter. We investigated whether neutralisation of free radicals in cigarette filters is feasible and accompanied by a reduction in smoke toxicity. Addition of the bioflavonoid pine bark extract Pycnogenolfi to cigarette filters depleted free radicals in a dose dependent manner. This was paralleled by a reduction of toxicity and mutagenicity in rodent test models. In this model system, the acute toxicity of cigarette smoke was markedly reduced by up to 70% in rodents with 0.4 mg Pycnogenolfi in filters. Chronic exposure to cigarette smoke for 75 days revealed that Pycnogenolfifilters significantly reduced mutagenicity by up to 48% and decreased pathological changes in lung tissue.

Super-resolution dipole orientation mapping via polarization demodulation

Fluorescence polarization microscopy (FPM) aims to detect the dipole orientation of fluorophores and to resolve structural information for labeled organelles via wide-field or confocal microscopy. Conventional FPM often suffers from the presence of a large number of molecules within the diffraction-limited volume, with averaged fluorescence polarization collected from a group of dipoles with different orientations. Here, we apply sparse deconvolution and least-squares estimation to fluorescence polarization modulation data and demonstrate a super-resolution dipole orientation mapping (SDOM) method that resolves the effective dipole orientation from a much smaller number of fluorescent molecules within a sub-diffraction focal area. We further apply this method to resolve structural details in both fixed and live cells. For the first time, we show that different borders of a dendritic spine neck exhibit a heterogeneous distribution of dipole orientation. Furthermore, we illustrate that the dipole is always perpendicular to the direction of actin filaments in mammalian kidney cells and radially distributed in the hourglass structure of the septin protein under specific labelling. The accuracy of the dipole orientation can be further mapped using the orientation uniform factor, which shows the superiority of SDOM compared with its wide-field counterpart as the number of molecules is decreased within the smaller focal area. Using the inherent feature of the orientation dipole, the SDOM technique, with its fast imaging speed (at sub-second scale), can be applied to a broad range of fluorescently labeled biological systems to simultaneously resolve the valuable dipole orientation information with super-resolution imaging.

Quantitative combination of natural anti-oxidants prevents metabolic syndrome by reducing oxidative stress

Insulin resistance and abdominal obesity are present in the majority of people with the metabolic syndrome. Antioxidant therapy might be a useful strategy for type 2 diabetes and other insulin-resistant states. The combination of vitamin C (Vc) and vitamin E has synthetic scavenging effect on free radicals and inhibition effect on lipid peroxidation. However, there are few studies about how to define the best combination of more than three anti-oxidants as it is difficult or impossible to test the anti-oxidant effect of the combination of every concentration of each ingredient experimentally. Here we present a math model, which is based on the classical Hill equation to determine the best combination, called Fixed Dose Combination (FDC), of several natural anti-oxidants, including Vc, green tea polyphenols (GTP) and grape seed extract proanthocyanidin (GSEP). Then we investigated the effects of FDC on oxidative stress, blood glucose and serum lipid levels in cultured 3T3-L1 adipocytes, high fat diet (HFD)-fed rats which serve as obesity model, and KK-ay mice as diabetic model. The level of serum malondialdehyde (MDA) in the treated rats was studied and Hematoxylin-Eosin (HE) staining or Oil red slices of liver and adipose tissue in the rats were examined as well. FDC shows excellent antioxidant and anti-glycation activity by attenuating lipid peroxidation. FDC determined in this investigation can become a potential solution to reduce obesity, to improve insulin sensitivity and be beneficial for the treatment of fat and diabetic patients. It is the first time to use the math model to determine the best ratio of three anti-oxidants, which can save much more time and chemical materials than traditional experimental method. This quantitative method represents a potentially new and useful strategy to screen all possible combinations of many natural anti-oxidants, therefore may help develop novel therapeutics with the potential to ameliorate the worldwide metabolic abnormalities.

Developing novel methods to image and visualize 3D genomes

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

A novel method to identify topological domains using Hi-C data

Over the last decade the 3C-based (Chromosome Conformation Capture, 3C) approaches have been developed to describe the frequency of chromatin interaction. The invention of Hi-C allows us to obtain genome-wide chromatin interaction map. However, it is challenging to develop efficient and robust analytical tools to interpret the Hi-C data. Here we present a new method called Clustering based Hi-C Domain Finder (CHDF), which is based on the difference of interaction intensity inside/outside domains, to identify Hi-C domains. We also compared CHDF with existing methods including Direction Index (DI) and HiCseg. CHDF can define more chromatin domains validated by higher resolution local chromatin structure data (Chromosome Conformation Capture Carbon Copy (5C) data). Using Hi-C data of lower sequencing depth, chromatin structure identified by CHDF is closer to that discovered by data of higher sequencing depth. Furthermore, the implement of CHDF is faster than the other two. Using CHDF, we are potentially able to discover more hints and clues about chromatin structural elements at domain level.

Toxicologic damage of gas phase cigarette smoke on cells and the protective effect of green tea polyphenols

A mouse fibroblast cell line, Chinese hamster lung V79 cell, was used to assess the toxicological effect of gas phase cigarette smoke (GPCS) on cells and the protective effect of green tea polyphenols (GTP). Exposure of the cultured V79 cells to GPCS decreased cell viability and caused lipid peroxidation of cell membrane as measured by thiobarbituric acid reaction. Electron spin resonance (ESR) spin trapping and spin labeling studies indicated that GPCS exposure could increase the fluidity in the polar surface of cell membrane, but the membrane fluidity in the hydrophobic region was not affected. The conformation of sulfhydryl binding sites on membrane was changed. GTP itself had no effect on the polar surface of membrane nor changed the conformation of sulfhydryl binding sites on membrane proteins in the concentration used in this experiment. Incubation of V79 cells with GTP aqueous solution before GPCS treatment could decrease cell death and lipid peroxidation and inhibit the changes of the biophysical properties of cell membrane induced by GPCS in a dose-dependent manner. Also GTP could scavenge the free radicals generated by GPCS. These results indicate that GPCS can induce lipid peroxidation and affect the biophysical properties of cell membrane, which may account for the toxicity of GPCS, and that GTP can prevent these changes possibly by scavenging the free radicals in GPCS and inhibiting the membrane lipid peroxidation.

Developing bioimaging and quantitative methods to study 3D genome

The recent advances in chromosome configuration capture (3C)-based series molecular methods and optical superresolution (SR) techniques offer powerful tools to investigate three dimensional (3D) genomic structure in prokaryotic and eukaryotic cell nucleus. In this review, we focus on the progress during the last decade in this exciting field. Here we at first introduce briefly genome organization at chromosome, domain and sub-domain level, respectively; then we provide a short introduction to various super-resolution microscopy techniques which can be employed to detect genome 3D structure. We also reviewed the progress of quantitative and visualization tools to evaluate and visualize chromatin interactions in 3D genome derived from Hi-C data. We end up with the discussion that imaging methods and 3C-based molecular methods are not mutually exclusive — actually they are complemental to each other and can be combined together to study 3D genome organization.

EPR study of the toxicological effects of gas-phase cigarette smoke and the protective effects of grape seed extract on the mitochondrial membrane

Electron paramagnetic resonance spin trapping and spin labeling techniques were used to study the effects of grape seed extract on the biophysical propertiers of the mitochondrial membrane exposed to gas-phase cigarette smoke. The spin trapping results indicated that grape seed extract can effectively scavenge free radicals in cigarette smoke and the lipid free radicals generated from the lipid peroxidation of mitochondrial membrane caused by gas-phase cigarette smoke. The spin labeling results showed that grape seed extract can protect the mitochondrial membrane from the toxicological damage caused by gas-phase cigarette smoke.