Clinical and Molecular Study of the NOG Gene in Families with Mandibular Micrognathism.

OBJECTIVES: Previous studies showed that noggin gene (NOG) sequence alterations, as well as epigenetic factors, could influence mandibular development. The aim of this study was to analyze clinical characteristics, NOG gene sequences, and promoter methylation sites in patients with mandibular micrognathism. MATERIALS AND METHODS: A total of 35 individuals of five Colombian families were subject to clinical and cephalometric analysis for mandibular micrognathism. One nonaffected individual of each family was included as a control. DNA was isolated from whole blood sample from all individuals by salting out method. Nine NOG gene fragments were amplified by polymerase chain reaction (PCR) and sequenced. Identification of CpG islands for methylation analysis at the NOG gene promoter was performed by MSP-PCR kit (Qiagen R). STATISTICAL ANALYSIS: A descriptive statistical analysis was carried out evaluating the presence or absence of genetics variants and the methylation sites in the NOG gene. RESULTS: NOG sequence results of affected individuals with mandibular micrognathism for one of the families studied demonstrated that they were heterozygous for 672 C/A (new mutation). For a second family, individuals were heterozygous for 567 G/C (single nucleotide polymorphism [SNP] RS116716909). For DNA analyzed from all patients studied, no methylations were observed at the NOG gene promoter region. CONCLUSION: Our results suggested that 672 C/A and 567 G/C variants could be involved in the presence of mandibular micrognathism. Moreover, lack of methylation sites at the NOG gene promoter region of all individuals studied suggests possibly other epigenetic factors could modulate mandibular growth. The search of genetic variants related with mandibular micrognathism will allow to predict in an integral way the development patterns of the patients and therefore establish a better clinical treatment.

Proteínas salivales relacionadas con la diversidad de dietas en murciélagos tropicales / Salivary proteins related to the diversity of diets in tropical bats

La familia Phyllostomidae presenta una gran diversidad de dietas que requieren adaptaciones fisiológicas para metabolizar los diferentes alimentos que consumen. En frugívoros de la familia Pteropodidae e insectívoros de las familias Vespertilionidae y Molossidae se han reportado proteínas salivales distintivas de cada dieta. Por ello, se planteó determinar moléculas salivales asociadas con las diferentes dietas de los filostómidos. Los organismos se encontraban en ayuno al tomar la muestra, a la cual se le adicionó un buffer inhibidor de proteasas y se almacenó a -20°C hasta su uso. Las proteínas se identificaron por medio de SDS-PAGE y se evaluó si su presencia en los individuos estaba asociada con la historia evolutiva de las especies. Además, se determinó si las proteínas encontradas estarían relacionadas con la dieta del individuo. Se capturaron 15 especies con dietas nectarívora, insectívora y frugívora. Se encontró una proteína de 60kDa en filostómidos herbívoros y una de 50kDa en vespertilionidos y filostómidos con alto consumo de insectos. Además, se registró una proteína de 30kDa en todos los filostómidos y en 2 de las 3 especies de vespertilionidos. Los análisis indicaron que la presencia de las proteínas no estaría relacionada con la cercanía filogenética y que, para las proteínas de 30 y 50kDa, tampoco sería explicada por la dieta como sí ocurre con la proteína de 60kDa. Los filostómidos habrían retenido de su dieta ancestral insectívora la proteína de 30kDa y adquirido evolutivamente la de 60kDa para procesar plantas y lograr la amplia diversificación ecológica que presentan.

The Phyllostomidae family presents a great diversity of diets that require physiological adaptations to metabolize the different foods that they consume. In frugivores of the family Pteropodidae and insectivores of the families Vespertilionidae and Molossidae, distinctive salivary proteins of each diet have been reported. For this reason, it was proposed to determine salivary molecules associated with the different diets of the phylostomids. The organisms were fasting when taking the sample, to which a protease inhibitor buffer was added and it was stored at -20°C until use. The proteins were identified by means of SDS-PAGE and it was evaluated whether their presence in individuals was associated with the evolutionary history of the species. In addition, it was determined whether the proteins found would be related to the individual's diet. 15 species were caught with nectarivorous, insectivorous and frugivorous diets. A protein of 60kDa was found in herbivorous phyllostomids and a 50kDa protein in vespertilionids and phyllostomids with high insect consumption. In addition, a 30kDa protein was recorded in all phylostomids and in 2 of the 3 species of vespertilionids. The analyzes indicated that the presence of the proteins would not be related to the phylogenetic closeness and that, for the 30 and 50kDa proteins, it would not be explained by the diet as it is the case with the 60kDa protein. The phylostomids would have retained the 30kDa protein from their ancestral insectivorous diet and evolutionarily acquired the 60kDa protein to process plants and achieve the broad ecological diversification they present.

time-ChIP: A Method to Determine Long-Term Locus-Specific Nucleosome Inheritance.

Understanding chromatin dynamics is essential to define the contribution of chromatin to heritable gene silencing and the long-term maintenance of gene expression. Here we present a detailed protocol for time-ChIP, a novel method to measure histone turnover at high resolution across long timescales. This method is based on the SNAP-tag, a self-labeling enzyme that can be pulse labeled with small molecules in cells. Upon pulse biotinylation of a cohort of SNAP-tagged histones we can determine their abundance and fate across a chase period using a biotin-specific chromatin pulldown followed by DNA sequencing or quantitative PCR. This method is unique in its ability to trace the long-term fate of a chromatin bound histone pool, genome wide. In addition to a step by step protocol, we outline advantages and limitations of the method in relation to other existing techniques. time-ChIP can define regions of high and low histone turnover and identify the location of pools of long lived histones.

Enhancer regions show high histone H3.3 turnover that changes during differentiation.

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation.

Basic properties of epigenetic systems: lessons from the centromere.

Chromatin-based epigenetic inheritance cooperates with cis-acting DNA sequence information to propagate gene expression states and chromosome architecture across cell division cycles. Histone proteins and their modifications are central components of epigenetic systems but how, and to what extent, they are propagated is a matter of continued debate. Centromeric nucleosomes, marked by the histone H3 variant CENP-A, are stable across mitotic divisions and are assembled in a locus specific and cell cycle controlled manner. The mechanism of inheritance of this unique chromatin domain has important implications for how general nucleosome transmission is controlled in space and time.