Biology subjects

Zhurkin, V. B.

Publications and source records attributed to Zhurkin, V. B..

8 recordsLinked to original sources

bioRxiv2026

BPabZIP, a new bZIP protein motif that promotes binding near, and displacement of, nucleosomes

Many transcription factors (TFs) bind only a subset of their canonical binding sites in mammalian cells. To identify differences between bound and unbound sites we examined Zta(N182S), a mutant of the Epstein Barr Virus (EBV)-encoded Zta bZIP protein that binds distinct DNA sequences that are not strongly bound by any known human or viral TF, reducing the effects of selective pressure on endogenous genomic binding sites. We stably expressed Zta(N182S) in human HEK293 cells and monitored protein binding (ChIP-seq) and effects on chromatin accessibility (ATAC-seq). Zta(N182S) binds ~10% of the 14,979 genomic occurrences of the canonical 9-mer ATCACTCAT, creating stronger overall ATAC-seq signal compared to control cells, suggesting nucleosome displacement. Nucleosome occupancy, either predicted or experimentally determined (MNase), indicates that canonical Zta and Zta(N182S) sites are more strongly bound when they are ~60bp from a positioned nucleosome dyad. These data suggest that Zta and Zta(N182S) binding results in nucleosome remodeling, consistent with pioneer-like activity. Examination of amino acids across Zta and human bZIPs identifies four conserved basic amino acids, a proline, and acidic amino acids immediately N-terminal of the basic amino acids of the bZIP domain (PARRTRKPQQPESLEECDSELEIKRYKN). We term this new protein motif "BPabZIP" (Basic-Proline-acidic bZIP). Molecular structure predictions for both Zta and human Fos/Jun reveal the basic amino acids interacting with the acidic patch on the nucleosome. The acidic amino acids act as an a-helical extension of the basic region that mimics DNA by interacting with histones H2A and H2B. Taken together, our analyses of this synthetic TF reveal a pioneer-like mechanism that is present in both human and viral bZIP proteins.

genomics↗

bioRxiv2023

Histone N-tails modulate sequence-specific positioning of nucleosomes

The precise mechanisms governing sequence-dependent positioning of nucleosomes on DNA remain unknown in detail. Existing algorithms, taking into account the sequence-dependent deformability of DNA and its interactions with the histone globular domains, predict rotational setting of only 65% of human nucleosomes mapped in vivo. To uncover novel factors responsible for the nucleosome positioning, we analyzed potential involvement of the histone N-tails in this process. To this aim, we reconstituted the H2A/H4 N-tailless nucleosomes on human BRCA1 DNA ([~]100 kb) and compared their positions and sequences with those of the wild-type nucleosomes. In the case of H2A tailless nucleosomes, the AT content of DNA sequences is changed locally at superhelical location (SHL) {+/-}4, while maintaining the same rotational setting as their wild-type counterparts. Conversely, the H4 tailless nucleosomes display widespread changes of the AT content near SHL {+/-}1 and SHL {+/-}2, where the H4 N-tails interact with DNA. Furthermore, a substantial number of H4 tailless nucleosomes exhibit rotational setting opposite to that of the wild-type nucleosomes. Thus, our findings strongly suggest that the histone N-tails are operative in selection of nucleosome positions, which may have wide-ranging implications for epigenetic modulation of chromatin states.

molecular biology↗

bioRxiv2023

Nucleosome reorganisation in breast cancer tissues

Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. Here we have generated high resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with [~]20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. In addition, tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. These effects were correlated with gene activity, DNA sequence repeats abundance, differential DNA methylation and binding of linker histone variants H1.4 and H1X. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient stratification and monitoring using liquid biopsies.

genomics↗

bioRxiv2022

The methyltransferases METTL7A and METTL7B confer resistance to thiol-based histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACis) are part of a growing class of epigenetic therapies used for the treatment of cancer. While elevated levels of the efflux pump P-gp are associated with in vitro resistance to romidepsin, this mechanism does not translate to the clinic. We developed a romidepsin-resistant cell line with a resistance mechanism independent of P-gp function that acts upstream of the deacetylation process. We found that expression of the methyltransferase METTL7A is necessary for resistance, and that expression of METTL7A in naive cells can drive resistance to thiol-containing HDACis. We demonstrate that METTL7A can methylate romidesin in vitro and that the ability of METTL7A to drive resistance to thiol-containing HDACis can be blocked by the methyltransferase inhibitor DCMB. Our data supports a model whereby exposure of cells to romidepsin selects for upregulation of the methyltransferase METTL7A, which in turn modifies the zinc-binding thiol, inactivating the drug.

cancer biology↗

bioRxiv2019

CTCF binding strength modulates chromatin architecture through the changes of the local nucleosome repeat length

The CCCTC-binding factor (CTCF) organises the genome in 3D through DNA loops and in 1D by setting boundaries isolating different chromatin states, but these processes are not well understood. Here we focus on the relationship between CTCF binding and the decrease of the Nucleosome Repeat Length (NRL) for [~]20 adjacent nucleosomes, affecting up to 10% of the mouse genome. We found that the chromatin boundary near CTCF is created by the nucleosome-depleted region (NDR) asymmetrically located >40 nucleotides 5-upstream from the centre of CTCF motif. The strength of CTCF binding to DNA is correlated with the decrease of NRL near CTCF and anti-correlated with the level of asymmetry of the nucleosome array. Individual chromatin remodellers have different contributions, with Snf2h having the strongest effect on the NRL decrease near CTCF and Chd4 playing a major role in the symmetry breaking. Upon differentiation of embryonic stem cells to neural progenitor cells and embryonic fibroblasts, a subset of common CTCF sites preserved in all three cell types maintains a relatively small local NRL despite genome-wide NRL increase. The sites which lost CTCF upon differentiation are characterised by nucleosome rearrangement 3-downstream, but the boundary defined by the NDR 5-upstream of CTCF motif remains.

genomics↗

bioRxiv2018

Polymorphic 30-nm Chromatin Fiber and Linking Number Paradox. Evidence for the Occurrence of Two Distinct Topoisomers.

We discuss various models of spatial organization of the 30-nm fiber that remains enigmatic despite 40 years of intensive studies. Using computer simulations, we found two topologically different families of the fiber conformations distinguished by the linker length, L: the fibers with L = {10n} and {10n+5} bp have DNA linking numbers per nucleosome DLk >> -1.5 and -1.0, respectively. The fibers with DLk >> -1.5 were observed earlier, while the topoisomer with DLk >> -1.0 is novel. These predictions were confirmed for circular nucleosome arrays with precisely positioned nucleosomes. We suggest that topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. In particular, the {10n+5} DNA linkers are likely to produce transcriptionally competent chromatin structures. This hypothesis is consistent with available data for several eukaryotes, from yeast to human. We also analyzed two recent studies of chromatin fibers - on the nucleosome crosslinking in vitro, and on radioprobing DNA folding in human cells. In both cases, we show that the novel topoisomer with DLk >> -1.0 has to be taken into account to interpret experimental data. This is yet another evidence for occurrence of two distinct fiber topoisomers. Potentially, our findings may reflect a general tendency of chromosomal domains with different levels of transcription to retain topologically different higher-order structures.

molecular biology↗

bioRxiv2017

p53 binding sites in normal and cancer cells are characterized by distinct chromatin context

The tumor suppressor protein p53 interacts with DNA in a sequence-dependent manner. Thousands of p53 binding sites have been mapped genome-wide in normal and cancer cells. However, the way p53 selectively binds its cognate sites in different types of cells is not fully understood. Here, we performed a comprehensive analysis of 25 published p53 cistromes and identified 3,551 and 6,039 high-confidence binding sites in normal and cancer cells, respectively. Our analysis revealed two distinct epigenetic features underlying p53-DNA interactions in vivo. First, p53 binding sites are associated with transcriptionally active histone marks (H3K4me3 and H3K36me3) in normal-cell chromatin, but with repressive histone marks (H3K27me3) in cancer-cell chromatin. Second, p53 binding sites in cancer cells are characterized by a lower level of DNA methylation than their counterparts in normal cells, probably related to global hypomethylation in cancers. Intriguingly, regardless of the cell type, p53 sites are highly enriched in the endogenous retroviral elements of the ERV1 family, highlighting the importance of this repeat family in shaping the transcriptional network of p53. Moreover, the p53 sites exhibit an unusual combination of chromatin patterns: high nucleosome occupancy and, at the same time, high sensitivity to DNase I. Our results suggest that p53 can access its target sites in a chromatin environment that is non-permissive to most DNA-binding transcription factors, which may allow p53 to act as a pioneer transcription factor in the context of chromatin.

genomics↗

bioRxiv2017

DNA topology in chromatin is defined by nucleosome spacing

In eukaryotic chromatin, DNA makes about 1.7 left superhelical turns around an octamer of core histones implying that formation of nucleosomes would alter the overall topology of DNA by a comparable difference of the DNA linking number ({Delta}Lk) per nucleosome. However, earlier experiments have documented a significantly (about 50%) lower absolute value |{Delta}Lk| than expected from the nucleosome geometry. Recently, using computer modeling, we have predicted two families of energetically stable conformations of the arrays with precisely positioned nucleosomes, one with an integer number of DNA turns in the linker DNA {L = 10n} and the other with extra five base pairs in the linker {L = 10n + 5}, to be topologically different. Here, using arrays of precisely positioned clone 601 nucleosomes, topological electrophoretic assays, and electron microscopy we experimentally tested these predictions. First, for small 12-mer nucleosome circular arrays we observed that dLk per nucleosome changes from -1.4 to -0.9 for the linkers {L = 10n} and {L = 10n + 5}, respectively. Second, for larger hybrid arrays containing a mixture of positioned and non-positioned nucleosomes we found that changing the DNA linker length within the positioned arrays was sufficient to significantly alter the overall DNA topology fully consistent with our prediction. The observed topological polymorphism of the circular nucleosome arrays provides a simple explanation for the DNA topology in native chromatin with variable DNA linker length. Furthermore, our results may reflect a more general tendency of chromosomal domains containing active or repressed genes to acquire different nucleosome spacing to retain topologically distinct higher-order structures.

molecular biology↗