Xét nghiệm phân mảnh DNA tinh trùng và khuyến cáo trong thực hành lâm sàng

Tinh dịch đồ là xét nghiệm đầu tay đánh giá khả năng sinh sản nam giới, nhưng xét nghiệm này không thể phản ánh chính xác những biến đổi vật chất di truyền trong nhân tinh trùng, cũng như không thể tiên lượng được kết cục điều trị trong hỗ trợ sinh sản. Tính toàn vẹn DNA tinh trùng đóng vai trò quan trọng cho sự phát triển của phôi cũng như là dấu hiệu sinh học đại diện cho một tinh trùng khỏe mạnh. Do đó, các kỹ thuật xét nghiệm phân mảnh DNA tinh trùng ngày càng được thực hiện phổ biến. Hiện nay, một số kỹ thuật thường được sử dụng để đánh giá phân mảnh DNA tinh trùng bao gồm TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), Comet (Single cell gel electrophore sis), SCD (Sperm chromatin dispersion) và SCSA (Sperm chromatin structure assay). Cho đến nay, vẫn chưa có khuyến cáo cụ thể cho chỉ định thực hiện xét nghiệm phân mảnh DNA tinh trùng. Bài tổng quan nhằm giới thiệu về các kỹ thuật xét nghiệm phân mảnh DNA tinh trùng cũng như tổng hợp khuyến cáo cho chỉ định thực hiện xét nghiệm này.

High affinity enhancer-promoter interactions can bypass CTCF/cohesin-mediated insulation and contribute to phenotypic robustness

Transcriptional control by distal enhancers is an integral feature of gene regulation. To understand how enhancer-promoter interactions arise and assess the impact of disrupting 3D chromatin structure on gene expression, we generated an allelic series of mouse mutants that perturb the physical structure of the Sox2 locus. We show that in the epiblast and in neuronal tissues, CTCF-mediated loops are neither required for the interaction of the Sox2 promoter with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs between Sox2 and its distal enhancers generated ectopic loops with varying degrees of insulation that directly correlated with reduced transcriptional output. Yet, even the mutants exhibiting the strongest insulation, with six CTCF motifs in divergent orientation, could not fully abolish activation by distal enhancers, and failed to disrupt implantation and neurogenesis. In contrast, cells of the anterior foregut were more susceptible to chromatin structure disruption with no detectable SOX2 expression in mutants with the strongest CTCF-mediated boundaries. These animals phenocopied loss of SOX2 in the anterior foregut, failed to separate trachea from esophagus and died perinatally. We propose that baseline transcription levels and enhancer density may influence the tissue-specific ability of distal enhancers to overcome physical barriers and maintain faithful gene expression. Our work suggests that high affinity enhancer-promoter interactions that can overcome chromosomal structural perturbations, play an essential role in maintaining phenotypic robustness.

Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells

The three-dimensional configuration of the genome ensures cell type-specific gene expression profiles by placing genes and regulatory elements in close spatial proximity. Here, we used in situ high-throughput chromosome conformation (in situ Hi-C), RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to characterize the high-order chromatin structure signature of female germline stem cells (FGSCs) and identify its regulating key factor based on the data-driven of multiple omics data. By comparison with pluripotent stem cells (PSCs), adult stem cells (ASCs), and somatic cells at three major levels of chromatin architecture, A/B compartments, topologically associating domains, and chromatin loops, the chromatin architecture of FGSCs was most similar to that of other ASCs and largely different from that of PSCs and somatic cells. After integrative analysis of the three-dimensional chromatin structure, active compartment-associating loops (aCALs) were identified as a signature of high-order chromatin organization in FGSCs, which revealed that CCCTC-binding factor was a major factor to maintain the properties of FGSCs through regulation of aCALs. We found FGSCs belong to ASCs at chromatin structure level and characterized aCALs as the high-order chromatin structure signature of FGSCs. Furthermore, CTCF was identified to play a key role in regulating aCALS to maintain the biological functions of FGSCs. These data provide a valuable resource for future studies of the features of chromatin organization in mammalian stem cells and further understanding of the fundamental characteristics of FGSCs.

Determination of Slow-binding HDAC Inhibitor Potency and Subclass Selectivity

Histone deacetylases (HDACs) 1-3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1-3 often display slow-binding kinetics, which causes a delay in inhibitor-enzyme equilibration and may affect assay readout. Here, we compare the potency and selectivity of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1-3 by a two-step, slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1-3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.

Modeling the Three-Dimensional Chromatin Structure from Hi-C Data with Transfer Learning

Recent studies have revealed the importance of three-dimensional (3D) chromatin structure in the regulation of vital biological processes. Contrary to protein folding, no experimental procedure that can directly determine ground-truth 3D chromatin coordinates exists. Instead, chromatin conformation is studied implicitly using high-throughput chromosome conformation capture (Hi-C) methods that quantify the frequency of all pairwise chromatin contacts. Computational methods that infer the 3D chromatin structure from Hi-C data are thus unsupervised, and limited by the assumption that contact frequency determines Euclidean distance. Inspired by recent developments in deep learning, in this work we explore the idea of transfer learning to address the crucial lack of ground-truth data for 3D chromatin structure inference. We present a novel method, Transfer learning Encoder for CHromatin 3D structure prediction (TECH-3D) that combines transfer learning with creative data generation procedures to reconstruct chromatin structure. Our work outperforms previous deep learning attempts for chromatin structure inference and exhibits similar results as state-of-the-art algorithms on many tests, without making any assumptions on the relationship between contact frequencies and Euclidean distances. Above all, TECH-3D presents a highly creative and novel approach, paving the way for future deep learning models.

Black chromatin is indispensable for accurate simulations of Drosophila melanogaster chromatin structure.

The interphase chromatin structure is extremely complex, precise and dynamic. Experimental methods can only show the frequency of interaction of the various parts of the chromatin. Therefore, it is extremely important to develop theoretical methods to predict the chromatin structure. In this publication, we describe the necessary factors for the effective modeling of the chromatin structure in Drosophila melanogaster. We also compared Monte Carlo with Molecular Dynamic methods. We showed that incorporating black, non-reactive chromatin is necessary for successfully prediction of chromatin structure, while the loop extrusion model or using Hi-C data as input are not essential for the basic structure reconstruction.

Kidney cell type specific changes in the chromatin and transcriptome landscapes following epithelial Hdac1and Hdac2 knockdown

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase-Accessible Chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24-30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.

Phasor Histone FLIM-FRET Microscopy Maps Nuclear-Wide Nanoscale Chromatin Architecture With Respect to Genetically Induced DNA Double-Strand Breaks

A DNA double-strand break (DSB) takes place in the context of chromatin, and there is increasing evidence for chromatin structure to play a functional role in DSB signaling and repair. Thus, there is an emerging need for quantitative microscopy methods that can directly measure chromatin network architecture and detect changes in this structural framework upon DSB induction within an intact nucleus. To address this demand, here we present the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labeled histones in the DSB inducible via AsiSI cell system (DIvA), which has sufficient spatial resolution to map nuclear-wide chromatin compaction at the level of nucleosome proximity with respect to multiple site-specific DSBs. We also demonstrate that when phasor histone FLIM-FRET is coupled with immunofluorescence, this technology has the unique advantage of enabling exploration of any heterogeneity that exists in chromatin structure at the spatially distinct and genetically induced DSBs.

Linker histone H1FOO is required for bovine preimplantation development by regulating lineage specification and nucleosome assembly

Linker histone H1 binds to the nucleosome and is implicated in the regulation of the chromatin structure and function. The H1 variant H1FOO is heavily expressed in oocytes and early embryos. However, given the poor homology of H1FOO among mammals, the functional role of H1FOO during early embryonic development remains largely unknown, especially in domestic animals. Here, we find that H1FOO is not only expressed in oocytes and early embryos but granulosa cells and spermatids in cattle. We then demonstrate that the interference of H1FOO results in early embryonic developmental arrest in cattle using either RNA editing or Trim-Away approach. H1FOO depletion leads to compromised expression of critical lineage-specific genes at the morula stage and affects the establishment of cell polarity. Interestingly, H1FOO depletion causes a significant increase in expression genes encoding other linker H1 and core histones. Concurrently, there is an increase of H3K9me3 and H3K27me3, two markers of repressive chromatin and a decrease of H4K16ac, a marker of open chromatin. Importantly, overexpression of bovine H1FOO results in severe embryonic developmental defects. In sum, we propose that H1FOO controls the proper chromatin structure that is crucial for the fidelity of cell polarization and lineage specification during bovine early development.