Search Results (18)

Codon optimization is a widely employed strategy to enhance protein expression. However, it occasionally leads to unexpected transcriptional repression despite preserving amino acid sequences. This study investigates the mechanistic basis of such transcriptional attenuation by analyzing two gene candidates (0432 and Fluc) in the common expression chassis P. pastoris. Both genes experienced severe mRNA reduction following codon optimization. Evidenced by histone H3 chromatin immunoprecipitation (ChIP) and a DNase I hypersensitivity assay, gene sequences with transcriptional repression displayed elevated nucleosome occupancy and reduced chromatin accessibility. The above change was caused by an ORF sequence change independent of the promoter, since transcriptional attenuation and compromised chromatin accessibility were still observed after replacing the strong promoter P_GAP with P_por1 or P_rps8b. Our findings challenge the conventional view of codon optimization as solely translation-centric, revealing its capacity to preemptively modulate transcription through chromatin accessibility. This work underscores the necessity of integrating chromatin-level considerations into synthetic gene design to avoid unintended transcriptional silencing and optimize expression outcomes. Full article

Wheat powdery mildew disease caused by the obligate biotrophic fungal pathogen Blumeria graminis forma specialis tritici (B.g. tritici) seriously threatens global wheat production. Although improved powdery mildew resistance is an aim in wheat breeding, the regulatory mechanism underlying the wheat–B.g. tritici interaction remains poorly understood. In this study, the wheat chromatin remodeling protein TaSWP73 was identified as a negative regulator of post-penetration resistance against B.g. tritici. The transient overexpression of TaSWP73 attenuates wheat post-penetration resistance against B.g. tritici, while the silencing of TaSWP73 potentiates salicylic acid (SA) biosynthesis and activates post-penetration resistance against B.g. tritici. Importantly, chromatin in the promoter regions of TaSARD1, an activator gene of SA biosynthesis, is marked by high nucleosome occupancy in the TaSWP73-silenced wheat leaves. The silencing of TaSARD1 could suppress SA biosynthesis and attenuate post-penetration resistance against B.g. tritici with a lack of TaSWP73. In addition, TaICS1 was characterized as an essential component of wheat SA biosynthetic machinery. Potentiated SA biosynthesis and increased post-penetration resistance against B.g. tritici with a lack of TaSWP73 could be suppressed by the silencing of TaICS1 expression. These results collectively support the hypothesis that the wheat chromatin remodeling protein TaSWP73 contributes to the compatible wheat–powdery mildew interaction presumably via the suppression of the TaSARD1-TaICS1-SA pathway. Full article

(1) Background: Nucleosomes represent the essential structural units of chromatin and serve as key regulators of cell function and gene expression. Oocytes in the germinal vesicle (GV) stage will later undergo meiosis and become haploid cells ready for fertilization, while somatic cells undergo mitosis after DNA replication. (2) Purpose: To furnish theoretical insights and data that support the process of cell reprogramming after nuclear transplantation. (3) Methods: We compared the nucleosome occupancy, distribution, and transcription of genes between two types of cells: fully grown GV oocytes from big follicles (BF) and somatic cells (porcine embryonic fibroblast, PEF). (4) Results: The nucleosome occupancy in the promoter of BF was 4.85%, which was significantly higher than that of 3.3% in PEF (p < 0.05), and the nucleosome distribution showed a noticeable increase surrounding transcriptional start sites (TSSs) in BF. Next, we reanalyzed the currently published transcriptome of fully grown GV oocytes and PEF, and a total of 51 genes in BF and 80 genes in PEF were identified as being uniquely expressed. The nucleosome distribution around gene TSSs correlated with expression levels in somatic cells, yet the results in BF differed from those in PEF. (5) Conclusion: This study uncovers the dynamic nature and significance of nucleosome positioning and chromatin organization across various cell types, providing a basis for nuclear transplantation. Full article

Efficient transcriptional regulation of the stress response is critical for microorganism survival. In yeast, stress-related gene expression, particularly for antioxidant enzymes like catalases, mitigates reactive oxygen species such as hydrogen peroxide (H₂O₂), preventing cell damage. The halotolerant yeast Debaryomyces hansenii shows oxidative stress tolerance, largely due to high catalase activity from DhCTA and DhCTT genes. This study evaluates D. hansenii’s response to oxidative stress caused by H₂O₂ under saline conditions, focusing on cell viability, gene expression, and catalase activity. Chromatin organization in the promoter of DhCTA and DhCTT was analyzed, revealing low nucleosome occupancy in promoter regions, correlating with active gene expression. Stress-related motifs for transcription factors like Msn2/4 and Sko1 were found, suggesting regulation by the DhHog1 MAP kinase. Analysis of a Dhhog1Δ mutant showed DhHog1’s role in DhCTA expression under H₂O₂ or NaCl conditions. These findings highlight DhHog1’s critical role in regulating the stress response in D. hansenii, offering insights for enhancing stress tolerance in halotolerant yeasts, particularly for industrial applications in saline wastewater management. Full article

As the global population experiences a notable surge in aging demographics, the need to understand the intricate molecular pathways exacerbated by age-related stresses, including epigenetic dysregulation, becomes a priority. Epigenetic mechanisms play a critical role in driving age-related diseases through altered gene expression, genomic instability, and irregular chromatin remodeling. In this review, we focus on histones, a central component of the epigenome, and consolidate the key findings of histone loss and genome-wide redistribution as fundamental processes contributing to aging and senescence. The review provides insights into novel histone expression profiles, nucleosome occupancy, disruptions in higher-order chromatin architecture, and the emergence of noncanonical histone variants in the aging cellular landscape. Furthermore, we explore the current state of our understanding of the molecular mechanisms of histone deficiency in aging cells. Specific emphasis is placed on highlighting histone degradation pathways in the cell and studies that have explored potential strategies to mitigate histone loss or restore histone levels in aging cells. Finally, in addressing future perspectives, the insights gained from this review hold profound implications for advancing strategies that actively intervene in modulating histone expression profiles in the context of cellular aging and identifying potential therapeutic targets for alleviating a multitude of age-related diseases. Full article

In vitro-fertilized (IVF) and parthenogenetically activated (PA) embryos, key to genetic engineering, face more developmental challenges than in vivo-developed embryos (IVV). We analyzed single-cell RNA-seq data from the oocyte to eight-cell stages in IVV, IVF, and PA porcine embryos, focusing on developmental differences during early zygotic genome activation (ZGA), a vital stage for embryonic development. (1) Our findings reveal that in vitro embryos (IVF and PA) exhibit more similar developmental trajectories compared to IVV embryos, with PA embryos showing the least gene diversity at each stage. (2) Significant differences in maternal mRNA, particularly affecting mRNA splicing, energy metabolism, and chromatin remodeling, were observed. Key genes like SMARCB1 (in vivo) and SIRT1 (in vitro) played major roles, with HDAC1 (in vivo) and EZH2 (in vitro) likely central in their complexes. (3) Across different types of embryos, there was minimal overlap in gene upregulation during ZGA, with IVV embryos demonstrating more pronounced upregulation. During minor ZGA, global epigenetic modification patterns diverged and expanded further. Specifically, in IVV, genes, especially those linked to H4 acetylation and H2 ubiquitination, were more actively regulated compared to PA embryos, which showed an increase in H3 methylation. Additionally, both types displayed a distinction in DNA methylation. During major ZGA, IVV distinctively upregulated genes related to mitochondrial regulation, ATP synthesis, and oxidative phosphorylation. (4) Furthermore, disparities in mRNA degradation-related genes between in vivo and in vitro embryos were more pronounced during major ZGA. In IVV, there was significant maternal mRNA degradation. Maternal genes regulating phosphatase activity and cell junctions, highly expressed in both in vivo and in vitro embryos, were degraded in IVV in a timely manner but not in in vitro embryos. (5) Our analysis also highlighted a higher expression of many mitochondrially encoded genes in in vitro embryos, yet their nucleosome occupancy and the ATP8 expression were notably higher in IVV. Full article

Nucleosomes positioned on the HIV-1 5′ long terminal repeat (LTR) regulate sense transcription as well as the establishment and maintenance of latency. A negative-sense promoter (NSP) in the 3′ LTR expresses antisense transcripts with coding and non-coding activities. Previous studies identified cis-acting elements that modulate NSP activity. Here, we used the two chronically infected T cell lines, ACH-2 and J1.1, to investigate epigenetic regulation of NSP activity. We found that histones H3 and H4 are present on the 3′ LTR in both cell lines. Following treatment with histone deacetylase inhibitors (HDACi), the levels of H3K27Ac increased and histone occupancy declined. HDACi treatment also led to increased levels of RNA polymerase II (RNPII) at NSP, and antisense transcription was induced with similar kinetics and to a similar extent as 5′ LTR-driven sense transcription. We also detected H3K9me2 and H3K27me3 on NSP, along with the enzymes responsible for these epigenetic marks, namely G9a and EZH2, respectively. Treatment with their respective inhibitors had little or no effect on RNPII occupancy at the two LTRs, but it induced both sense and antisense transcription. Moreover, the increased expression of antisense transcripts in response to treatment with a panel of eleven latency-reversing agents closely paralleled and was often greater than the effect on sense transcripts. Thus, HIV-1 sense and antisense RNA expression are both regulated via acetylation and methylation of lysine 9 and 27 on histone H3. Since HIV-1 antisense transcripts act as non-coding RNAs promoting epigenetic silencing of the 5′ LTR, our results suggest that the limited efficacy of latency-reversing agents in the context of ‘shock and kill’ cure strategies may be due to concurrent induction of antisense transcripts thwarting their effect on sense transcription. Full article

Chromatin architecture is orchestrated, and plays crucial roles during the developmental process by regulating gene expression. In embryonic stem cells (ESCs), three types of chromatin states, including active, repressive and poised states, were previously identified and characterized with specific chromatin modification marks and different transcription activity, but it is largely unknown how nucleosomes are organized in these chromatin states. In this study, by using a DNA deformation energy model, we investigated the sequence-dependent nucleosome organization within the chromatin states in mouse ESCs. The results revealed that: (1) compared with poised genes, active genes are characterized with a higher level of nucleosome occupancy around their transcription start sites (TSS) and transcription termination sites (TTS), and both types of genes do not have a nucleosome-depleted region at their TTS, contrasting with the MNase-seq based result; (2) based on our previous DNA bending energy model, we developed an improved model capable of predicting both rotational positioning and nucleosome occupancy determined by a chemical mapping approach; (3) DNA bending-energy-based analyses demonstrated that the fragile nucleosomes positioned at both gene ends could be explained largely by enhanced rotational positioning signals encoded in DNA, but nucleosome phasing around the TSS of active genes was not determined by sequence preference; (4) the nucleosome occupancy landscape around the binding sites of some developmentally important transcription factors known to bind with different chromatin contexts, was also successfully predicted; (5) the difference of nucleosome occupancy around the TSS between CpG-rich and CpG-poor promoters was partly captured by our sequence-dependent model. Taken together, by developing an improved deformation-energy-based model, we revealed some sequence-dependent properties of the nucleosome arrangements in regions of distinct chromatin states in mouse ESCs. Full article

DNA damage is induced by exogenous and endogenous sources, creating a variety of lesions. However, the cellular repair machinery that addresses and corrects this damage must contend with the fact that genomic DNA is sequestered in the nucleoprotein complex of chromatin. As the minimal unit of DNA compaction, the nucleosome core particle (NCP) is a major determinant of repair and poses unique barriers to DNA accessibility. This review outlines how the base excision repair (BER) pathway is modulated by the NCP and describes the structural and dynamic factors that influence the ability of BER enzymes to find and repair damage. Structural characteristics of the NCP such as nucleobase positioning and occupancy will be explored along with factors that impact the dynamic nature of NCPs to increase mobilization of nucleosomal DNA. We will discuss how altering the dynamics of NCPs initiates a domino effect that results in the regulation of BER enzymes. Full article

All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease onset or pathological conditions that lead to increased cell death alter the contribution of different tissues to the total pool of cfDNA. Because cfDNA molecules retain cell-type specific epigenetic features, it is possible to infer tissue-of-origin from epigenetic characteristics. Recent research efforts demonstrated that analysis of, e.g., methylation patterns, nucleosome occupancy, and fragmentomics determined the cell- or tissue-of-origin of individual cfDNA molecules. This novel tissue-of origin-analysis enables to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies towards a wide range of pathologies and early diagnosis. In this review, we summarize the currently available tissue-of-origin approaches and point out the next steps towards clinical implementation. Full article

Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2′-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in Arabidopsis thaliana (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation. Full article

Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle–dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity. Full article

The transcriptome of every cell is orchestrated by the complex network of interaction between transcription factors (TFs) and their binding sites on DNA. Disruption of this network can result in many forms of organism malfunction but also can be the substrate of positive natural selection. However, understanding the specific determinants of each of these individual TF-DNA interactions is a challenging task as it requires integrating the multiple possible mechanisms by which a given TF ends up interacting with a specific genomic region. These mechanisms include DNA motif preferences, which can be determined by nucleotide sequence but also by DNA’s shape; post-translational modifications of the TF, such as phosphorylation; and dimerization partners and co-factors, which can mediate multiple forms of direct or indirect cooperative binding. Binding can also be affected by epigenetic modifications of putative target regions, including DNA methylation and nucleosome occupancy. In this review, we describe how all these mechanisms have a role and crosstalk in one specific family of TFs, the basic helix-loop-helix (bHLH), with a very conserved DNA binding domain and a similar DNA preferred motif, the E-box. Here, we compile and discuss a rich catalog of strategies used by bHLH to acquire TF-specific genome-wide landscapes of binding sites. Full article

The highly complex life cycle of the human malaria parasite, Plasmodium falciparum, is based on an orchestrated and tightly regulated gene expression program. In general, eukaryotic transcription regulation is determined by a combination of sequence-specific transcription factors binding to regulatory DNA elements and the packaging of DNA into chromatin as an additional layer. The accessibility of regulatory DNA elements is controlled by the nucleosome occupancy and changes of their positions by an active process called nucleosome remodeling. These epigenetic mechanisms are poorly explored in P. falciparum. The parasite genome is characterized by an extraordinarily high AT-content and the distinct architecture of functional elements, and chromatin-related proteins also exhibit high sequence divergence compared to other eukaryotes. Together with the distinct biochemical properties of nucleosomes, these features suggest substantial differences in chromatin-dependent regulation. Here, we highlight the peculiarities of epigenetic mechanisms in P. falciparum, addressing chromatin structure and dynamics with respect to their impact on transcriptional control. We focus on the specialized chromatin remodeling enzymes and discuss their essential function in P. falciparum gene regulation. Full article

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains. Full article