Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming.

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads. Stem Cells 2016;34:2418-2428.

Epigenetic reprogramming of mouse germ cells toward totipotency.

Primordial germ cells (PGCs), the precursors of sperm and eggs, are the route to totipotency and require establishment of a unique epigenome in this lineage. The genetic program for PGC specification in the mouse also initiates epigenetic reprogramming that continues when PGCs migrate into the developing gonads. Among these later events is active and genome-wide DNA demethylation, which is linked to extensive chromatin remodeling. These extensive epigenetic changes erase most, if not all, of the existing epigenetic information, which resets the epigenome for totipotency. Recent evidence suggests that active DNA demethylation involves a base excision repair (BER) pathway. BER is mechanistically linked to DNA demethylation, but what triggers BER is currently under investigation. The methylated cytosine (5mC) could be modified by deamination or to 5hmC, which could induce BER. Detection of Tet1 expression specifically and coincidentally, at the time of BER in PGCs, suggests that conversion of 5mC to 5hmC might be involved, at least in part, during epigenetic reprogramming and DNA demethylation in germ cells.

Germ line, stem cells, and epigenetic reprogramming.

The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.

The continuing quest to comprehend genomic imprinting.

The discovery of the phenomenon of genomic imprinting in mammals showed that the parental genomes are functionally non-equivalent. Considerable advances have occurred in the field over the past 20 years, which has resulted in the identification and functional analysis of a number of imprinted genes the expression of which is determined by their parental origin. These genes belong to many diverse categories and they have been shown to regulate growth, complex aspects of mammalian physiology and behavior. Many aspects of the mechanism of imprinting have also been elucidated. However, the reasons for the evolution of genomic imprinting remain enigmatic. Further research is needed to determine if there is any relationship between the apparently diverse functions of imprinted genes in mammals, and their role in human diseases. It also remains to be seen what common features exist amongst the diverse imprinting control elements. The mechanisms involved in the erasure and re-establishment of imprints should provide deeper insights into epigenetic mechanisms of wide general interest.

[Pulmonary veins and atrial fibrillation: a pathological study of 100 hearts]. / Plicní zíly a fibrilace síní - studie 100 srdcí.

Pathogenesis of atrial fibrillation (AF), the most common sustained heart arrhythmia, is not yet fully elucidated. Recent electrophysiological studies have shown that in most patients with AF the arrhythmia is triggered by ectopic beats originating from extensions of left atrial myocardium over the pulmonary veins (PVs), so called myocardial sleeves (MSPV). A total of 100 hearts (393 PVs) obtained at autopsy were prospectively studied - 50 from patients with chronic AF (average age 76.9 +/- 7.3 yrs.) and a control group of 50 with a sinus rhythm (aver. age 71.7 +/- 9.5 yrs.). This is a largest study published on this topic so far. It appeared that MSPV frequently harbour pathological lesions, particularly senile atrial amyloid, and scarring. These two pathological changes were evaluated semiquantitatively on a grade 0-3 basis in individual PVs, comparing the results in the AF vs. the control group. Amyloidosis of MSPV was found in 68 % of all hearts and in 55 % of all sleeves. The deposits were most marked in the right superior PV. Amyloidosis was more frequent and more severe in MSPV of patients with AF (58.5 %; average grade 0.89) than of those without AF (51.7 %; aver. grade 0.76); the differences, however, lack statistical significance. Scarring of MSPV was present in all 349 sleeves, more markedly in the left inferior, left superior, and right superior PVs. It was significantly more severe in patients with AF compared to those without the arrhythmia. By an injection metod, we have shown that MSPV are supplied by coronary arteries. However, the degree of scarring of the sleeves did not correlate with the degree of coronary atherosclerosis. We suggest that genesis of the scarring is not postnecrotic but degenerative, due to diffuse hypoxia of the sleeve myocardium. To conclude, amyloidosis and particularly scarring of MSPV appear generally in the elderly population as an arrhythmogenic substrate for AF.

[Study of the strength of compacts of mixed dry binders consisting of powdered cellulose and directly compressible lactose]. / Studium pevnosti výlisku ze smesných suchých pojiv slozených z práskové celulosy a prímo lisovatelné laktosy.

The paper studied the strength of compacts of dry binders consisting of powdered cellulose and directly compressible lactose. The powdered cellulose employed was Arbocel A300, the directly compressible lactose, Pharmatosa DCL 21. The first step of the evaluation comprised the tensile strength of compacts and sensitivity of dry binders alone to an addition of magnesium stearate. The same method of evaluation was then used for mixed dry binders from Arbocel A300 and Pharmatosa DCL 21 in ratios of 3:1, 1:1 and 1:3. The tested concentrations of magnesium stearate were 0.4 and 0.8%. Sensitivity of dry binders to an addition of the lubricant was evaluated by means of lubricant sensitivity ratio (LSR) values. The compacts with the highest strength and at the same time the lowest sensitivity to an addition of magnesium stearate were produced using a mixture of Arbocel A300 and Pharmatosa DCL 21 in a ratio of 1:3. The evaluation also included the commercially produced mixed dry binder Cellactosa 80, in which higher sensitivity to an addition of stearate than in a mixture of Arbocel A300 and Pharmatosa DCL 21 in a ratio of 1:3 was found.

CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression.

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

Bisulfite-based methylation analysis of imprinted genes.

Genomic imprinting is an epigenetically controlled form of gene regulation leading to the preferential expression of one parental gene copy. To date, approximately 40 imprinted genes have been described that are exclusively or predominantly expressed from either the paternal or the maternal allele (www.mgu.har.mrc.ac.uk/imprinting/implink.html). Changes in the imprinted expression of such genes result in developmental abnormalities; in the human they are associated with several diseases and various types of cancer (1-3).

Epidemiology of feline leukaemia and feline immunodeficiency virus infections in the Czech Republic.

Commercial serological sets were used for the examination of 727 cats kept in larger towns of the Czech Republic. FeLV antigen and antibodies to FIV were demonstrated in 96 (13.2%) and 42 (5.8%) of the animals, respectively. Seven (0.96%) animals were positive for both FeLV and FIV. Most of the FeLV and/or FIV positive patients were intact rambling males aged 1-4 years. Chronic gastrointestinal and respiratory diseases were found in 54.2% and 43.8% of the FeLV-positive patients, respectively. Chronic urinary tract diseases and generalized lymphadenopathy were found in 47.6% and 45.2% of the FIV-positive patients, respectively. The results of this first survey in the Czech Republic have shown prevalence values and clinical patterns similar to those reported formerly from other European countries.

The LTR, v-src, LTR provirus in H-19 hamster tumor cell line is integrated adjacent to the negative regulatory region.

The tumor hamster cell line H-19 harbors a single copy LTR, v-src, LTR provirus that becomes permanently transcriptionally suppressed in morphological revertants segregating at high rate from this cell line. Our previous data document that the provirus suppression is mediated by epigenetic cell-regulatory mechanisms. In this report, we concentrate on cellular sequences neighboring the integration site. The locus is unique for Syrian hamster and is not detectable in DNA of several animal species. No restriction sites that usually hint at the presence of CpG islands were found in the significantly close vicinity of the provirus. Nevertheless, the chromosomal DNA flanking the provirus is rich in GC content (57.8%). We localized a 0.5-kb region downstream from the provirus that remarkably inhibits transcription in the transient expression assay and is effective both on the homologous RSV LTR promoter/enhancer and heterologous SV40 promoter. We propose that a cellular trans-acting factor is involved in the silencing of the reporter gene. Since this activity is comparable both in transformed and revertant cells, we speculate that this down-regulatory region makes the permissive integration locus prone to provirus silencing initiated by other fluctuating stimuli.