Sergey V. Razin

Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition

Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin ‘ground state’ could potentially provide insights into reprogramming cells to a state of totipotency.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

Mapping of structural and transcription-related matrix attachment sites in the alpha-globin gene domain of avian erythroblasts and erythrocytes.

The positions of preferential DNA interaction with the nuclear matrix were mapped within the domain of the chicken alpha-globin genes in transcriptionally active erythroblast nuclei and inactive nuclei of mature erythrocytes. In the latter, only two major distinct attachment sites were observed, close to the A + T-rich sequences previously found at the boundaries of the domain. Sequencing of these structural matrix attachment points revealed several known DNA motifs; some of them were present on both sides of the domain. In actively transcribing erythroblast nuclei of adult animals, a large fraction of the transcribed area was represented in nuclear matrix DNA, including upstream and downstream elements. In particular, adult alpha A- and alpha D-globin genes were found in matrix DNA, while the transcribed but translationally unexpressed embryonic pi gene was underrepresented. The data are discussed in terms of the existence of stable or structural and expression-related matrix attachment sites; correlations to the origin of replication and the units of transcription of the domain are shown.

Cys2His2 zinc finger protein family: Classification, functions, and major members

Cys2His2 (C2H2)-type zinc fingers are widespread DNA binding motifs in eukaryotic transcription factors. Zinc fingers are short protein motifs composed of two or three β-layers and one α-helix. Two cysteine and two histidine residues located in certain positions bind zinc to stabilize the structure. Four other amino acid residues localized in specific positions in the N-terminal region of the α-helix participate in DNA binding by interacting with hydrogen donors and acceptors exposed in the DNA major groove. The number of zinc fingers in a single protein can vary over a wide range, thus enabling variability of target DNA sequences. Besides DNA binding, zinc fingers can also provide protein-protein and RNA-protein interactions. For the most part, proteins containing the C2H2-type zinc fingers are trans regulators of gene expression that play an important role in cellular processes such as development, differentiation, and suppression of malignant cell transformation (oncosuppression).

Chromosome Conformation Capture (from 3C to 5C) and Its ChIP-Based Modification

Chromosome conformation capture (3C) methodology was developed to study spatial organization of long genomic regions in living cells. Briefly, chromatin is fixed with formaldehyde in vivo to cross-link interacting sites, digested with a restriction enzyme and ligated at a low DNA concentration so that ligation between cross-linked fragments is favored over ligation between random fragments. Ligation products are then analyzed and quantified by PCR. So far, semi-quantitative PCR methods were widely used to estimate the ligation frequencies. However, it is often important to estimate the ligation frequencies more precisely which is only possible by using the real-time PCR. At the same time, it is equally necessary to monitor the specificity of PCR amplification. That is why the real-time PCR with TaqMan probes is becoming more and more popular in 3C studies. In this chapter, we describe the general protocol for 3C analysis with the subsequent estimation of ligation frequencies by using the real-time PCR technology with TaqMan probes. We discuss in details all steps of the experimental procedure paying special attention to weak points and possible ways to solve the problems. A special attention is also paid to the problems in interpretation of the results and necessary control experiments. Besides, in theory, we consider other approaches to analysis of the ligation products used in frames of the so-called 4C and 5C methods. The recently developed chromatin immunoprecipitation (ChIP)-loop assay representing a combination of 3C and ChIP is also discussed.

Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation—preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse β-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

Mapping of genomic DNA loop organization in a 500-kilobase region of the Drosophila X chromosome by the topoisomerase II-mediated DNA loop excision protocol.

The recently developed procedure of chromosomal DNA loop excision by topoisomerase II-mediated DNA cleavage at matrix attachment sites (S. V. Razin, R. Hancock, O. Iarovaia, O. Westergaard, I. Gromova, and G. P. Georgiev, Cold Spring Harbor Symp. Quant. Biol. 58:25-35, 1993; I. I. Gromova, B. Thompsen, and S. V. Razin, Proc. Natl. Acad. Sci. USA 92:102-106, 1995) has been employed for mapping the DNA loop anchorage sites in a 500-kb region of the Drosophila melanogaster X chromosome. Eleven anchorage sites delimiting 10 DNA loops ranging in size from 20 to 90 kb were found within this region. Ten of these 11 anchorage sites colocalize with previously mapped scaffold attachment regions. However, a number of other scaffold attachment regions are found to be located in loop DNA.

Mechanisms of heat shock response in mammals

Heat shock (HS) is one of the best-studied exogenous cellular stresses. The cellular response to HS utilizes ancient molecular networks that are based primarily on the action of stress-induced heat shock proteins and HS factors. However, in one way or another, all cellular compartments and metabolic processes are involved in such a response. In this review, we aimed to summarize the experimental data concerning all aspects of the HS response in mammalian cells, such as HS-induced structural and functional alterations of cell membranes, the cytoskeleton and cellular organelles; the associated pathways that result in different modes of cell death and cell cycle arrest; and the effects of HS on transcription, splicing, translation, DNA repair, and replication.

Nuclear matrix attachment regions and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin

We have mapped nuclear matrix attachment regions (MARs), defined by their specific binding to nuclear matrices in vitro, and sites of topoisomerase II reaction, detected by DNA cleavage in vitro in the presence of the inhibitor VM-26, in the vicinity of the replication origin of the chicken alpha-globin gene domain. Two MARs are located close to the downstream end (in the direction of transcription) of a 3 kb fragment which includes the origin. These MARs contain sites for strong topoisomerase II binding and reaction. Our observations on this gene domain support two hypotheses concerning MARs in eukaryotic cells, namely that they are close to DNA replication origins and that they contain multiple topoisomerase II recognition sites.

Mammalian recombination hot spot in a DNA loop anchorage region: A model for the study of common fragile sites

We analyzed the replication pattern and the topological organization of a 200 kb long Chinese hamster polygenic locus, which spans the boundary of two isochores. One of them is G + C rich while the second one is highly A + T rich. Previous analysis of mutants amplified for this locus had identified, within the A + T rich isochore, a mitotic recombination hotspot and a replication origin separated by some 7 kb. The recombination hotspot exhibits structural features repeatedly observed at common fragile sites, including a typical enrichment in peaks of enhanced DNA helix flexibility. By studying the replication pattern of the same locus in the non‐amplified CHO cells, we confirm here the localization of the replication origin and show that the mitotic recombination hotspot does not correspond to a replicon junction. This finding makes questionable current hypotheses correlating replication termination regions with recombination prone sequences. Using topoisomerase II‐mediated DNA cleavage at matrix attachment sites, we identified a 40 kb‐long DNA anchorage region extending all along a transcription unit nested within the A + T rich isochore. Both the recombination hotspot and the replication origin lie within this topoisomerase II sensitive region, which suggests that features essential for initiation of recombination and initiation of DNA replication cluster within DNA anchorage regions. Features common to this region and to common fragile sites are discussed. J. Cell. Biochem. Suppl. 36: 170–178, 2001.