Abstract
Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and ...extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.
The obesity pandemic has brought forth a scientific interest in food intake and sensory perception interactions. Olfactory perception and gustatory perception are very complex and under the influence ...of many factors, including the menstrual cycle. This study aims to clarify conflicting findings on the influence of the menstrual cycle on olfactory and gustatory perception. Women were assessed during four consecutive phases of one complete cycle (mid-follicular, ovulatory, mid-luteal, and late luteal phases (N = 21)), in contrast to women measured across the same phases belonging to two menstrual cycles (N = 29). Additional control groups were men (N = 17), postmenopausal women (N = 14), oral contraceptive users (N = 10), and women with an anovulatory cycle (N = 8). Olfactory threshold, odor discrimination, and identification were tested using the “Sniffin Sticks“ test kit. Suprathreshold intensity and hedonic ratings for sweet, salty, sour, and bitter solutions were assessed. One-way ANOVA and ANOVA for repeated measurements was applied in the analysis, along with linear and trigonometric data fitting and linear mixed models. Linear increases in olfactory discrimination, identification, and overall olfactory performance were observed only in women followed across a complete menstrual cycle. Compared to other groups, these women displayed a cyclic pattern characterized by a predilection for sweet solution; reduced distaste for salty and sour solutions; and increased intensity perception of salty, sour, and bitter solutions towards the end of the cycle. These results suggest that a distinct hormonal milieu of a complete menstrual cycle may be affecting both olfactory and gustatory perception.
Nuts are often considered beneficial for health, yet few studies have examined determinants of their intake and the associations between nut consumption and various cardiovascular disease risk ...factors. The aim of this study was to identify factors associated with nut intake in a Mediterranean population, in Croatia, and to investigate the association of nut intake and various cardiovascular risk factors.
Subjects from the Island of Vis, Island of Korčula and the City of Split were included in this cross-sectional study (
= 4416 in total; 4011 without known cardiovascular disease). Survey responses, medical records and clinically relevant measurements were utilized. Multivariate ordinal and logistic regression models were used in the analysis, adjusting for known confounding factors.
As low as 5% of all subjects reported daily, and 11% reported weekly, nut consumption. The characteristics associated with more frequent nut intake were female gender (Odds ratio (OR) = 1.39; 95% confidence interval (CI) 1.19-1.62), highest level of education (1.42; 1.15-1.76) and material status (1.58; 1.29-1.93), smoking abstinence (1.21; 1.04-1.42 in never-smokers and 1.22; 1.02-1.46 in ex-smokers), Mediterranean diet adherence (1.87; 1.62-2.15), and absence of central obesity (1.29; 1.09-1.53), absence of diabetes (1.30; 1.02-1.66) and metabolic syndrome (1.17; 1.01-1.36). Subjects who consumed nuts had more favorable waist-to-height (overall
= 0.036) and waist-to-hip ratios (0.033), lesser odds of elevated fibrinogen (
< 0.001 in both weekly and monthly nut consumers) and reduced high-density lipoprotein (HDL) cholesterol (
= 0.026), compared to non-consumers.
It appears that frequent nut consumption is an integral part of a healthy lifestyle and better socioeconomic status. A beneficial association of nut intake with cardiovascular risk factors was confirmed in this study.
Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present ...in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.
Chronic stress has various effects on organisms and is sex-specific. The aim of the study was to describe the expression of synapse strengthening protein, dendrin, in the spinal cord (SC) and the ...dependence of its expression on chronic stress and sex hormones. Thirteen-month-old female and male rats were castrated (ovariectomy F-OVX or orchidectomy M-ORX) or sham-operated (F-SH or M-SH), respectively. At age 15 months, three 10-day-sessions of sham stress (control, C) or chronic stress (S) were conducted. Dendrin expression was present in the thoracic SC segments and the dorsal root ganglia (DRG). In the SC, dendrin expression was prominent in superficial laminae of the dorsal horn and lamina X (central canal). The M-ORX-S group had the highest dendrin expression in the dorsal horn, being significantly higher than the M-ORX-C or M-SH-S groups (P < 0.05). Dendrin expression was significantly higher in the F-SH-S group than the F-SH-C group (P < 0.05), as well as in the F-SH-S than the M-SH-S (P < 0.05). Co-localization with the α-d-galactosyl-specific isolectin B4 (IB4) in central projections of the DRG neurons in the dorsal horn of the SC was 7.43 ± 3.36%, while with the calcitonin gene-related peptide (CGRP) was 8.47 ± 4.45%. Localization of dendrin was observed in soma and nuclei of neurons in the dorsal horn. Dendrin expression in pain-processing areas of the SC, the DRG neurons and their peripheral projections suggest possible roles in pain perception and modulation. Stress-induced increase in dendrin expression and its dependence on sex hormones may partially explain sex-specific pain hypersensitivity induced by stress.
Alternative glycosylation of immunoglobulin G (IgG) is functionally important in multiple human physiological and pathological states. Our understanding of molecular mechanisms that regulate IgG ...glycosylation is vague because of the complexity of this process, which involves hundreds of genes. Several genome-wide association (GWA) studies have revealed a network of genes associated with IgG glycosylation that are pleiotropic for a number of diseases. Here, we report a design of a versatile system for IgG production and gene manipulations that can be used for
in vitro
functional follow-up of GWA hits or any gene of interest. The system is based on CRISPR-dCas9, extended by a piggyBac integrase compatible vector, and drives IgG production in HEK-293F cells. We validated our systems that stably express VPR-dCas9 and KRAB-dCas9 by manipulation of four glyco-genes with a known role in IgG glycosylation, and then functionally validated three GWAS hits for IgG glycosylation with an as-yet-unknown role in this process.
After fertilization, to initiate development, gametes are reprogramed to become totipotent. Approximately half of the mammalian genome consists of repetitive elements, including retrotransposons, ...some of which are transcribed after fertilization. Retrotransposon activation is generally assumed to be a side effect of the extensive chromatin remodeling underlying the epigenetic reprogramming of gametes. Here, we used a targeted epigenomic approach to address whether specific retrotransposon families play a direct role in chromatin organization and developmental progression. We demonstrate that premature silencing of LINE-1 elements decreases chromatin accessibility, whereas prolonged activation prevents the gradual chromatin compaction that occurs naturally in developmental progression. Preventing LINE-1 activation and interfering with its silencing decreases developmental rates independently of the coding nature of the LINE-1 transcript, thus suggesting that LINE-1 functions primarily at the chromatin level. Our data suggest that activation of LINE-1 regulates global chromatin accessibility at the beginning of development and indicate that retrotransposon activation is integral to the developmental program.
Cellular plasticity is essential for early embryonic cells. Unlike pluripotent cells, which form embryonic tissues, totipotent cells can generate a complete organism including embryonic and ...extraembryonic tissues. Cells resembling 2-cell-stage embryos (2C-like cells) arise at very low frequency in embryonic stem (ES) cell cultures. Although induced reprogramming to pluripotency is well established, totipotent cells remain poorly characterized, and whether reprogramming to totipotency is possible is unknown. We show that mouse 2C-like cells can be induced in vitro through downregulation of the chromatin-assembly activity of CAF-1. Endogenous retroviruses and genes specific to 2-cell embryos are the highest-upregulated genes upon CAF-1 knockdown. Emerging 2C-like cells exhibit molecular characteristics of 2-cell embryos and higher reprogrammability than ES cells upon nuclear transfer. Our results suggest that early embryonic-like cells can be induced by modulating chromatin assembly and that atypical histone deposition may trigger the emergence of totipotent cells.
The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this ...plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.
The X-linked gene
plays major roles in female mouse development and reproduction, where it is crucial for the maintenance of imprinted X chromosome inactivation in extraembryonic tissues of embryos. ...However, while females carrying a systemic
knockout (KO) die around implantation, male
KO mice appear healthy and are fertile. Here, we report an important role for
in testis where it is highly expressed in post-meiotic round spermatids as well as in Sertoli cells. Systemic deletion of the
gene results in lower numbers of mature sperm that contains excess cytoplasm, leading to decreased sperm motility and in vitro fertilization rates. Targeting the conditional
cKO specifically to the spermatogenic cell lineage largely recapitulates this phenotype. These results reveal functions of
in male reproduction specifically in round spermatids during spermiogenesis.
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