Nucleosome Occupancy Research Articles

Differential modulation of polycomb-associated histone marks by cBAF, pBAF, and gBAF complexes.

Chromatin regulators alter the physical properties of chromatin to make it more or less permissive to transcription by modulating another protein's access to a specific DNA sequence through changes in nucleosome occupancy or histone modifications at a particular locus. Mammalian SWI/SNF complexes are a group of ATPase-dependent chromatin remodelers. In mouse embryonic stem cells, there are three primary forms of mSWI/SNF: canonical BAF (cBAF), polybromo-associated BAF (pBAF), and GLTSCR-associated BAF (gBAF). Nkx2-9 is bivalent, meaning nucleosomes at the locus have active and repressive modifications. In this study, we used unique BAF subunits to recruit each of the three complexes to Nkx2-9 using dCas9-mediated inducible recruitment (FIRE-Cas9). We show that recruitment of cBAF complexes leads to a significant loss of the polycomb repressive-2 H3K27me3 histone mark and polycomb repressive-1 and repressive-2 complex proteins, whereas gBAF and pBAF do not. Moreover, nucleosome occupancy alone cannot explain the loss of these marks. Our results demonstrate that cBAF has a unique role in the direct opposition of polycomb-associated histone modifications that gBAF and pBAF do not share.

Regulation of Catalase Expression and Activity by DhHog1 in the Halotolerant Yeast Debaryomyces hansenii Under Saline and Oxidative Conditions

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.

A repressive H3K36me2 reader mediates Polycomb silencing

In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.

Sir2 and Fun30 regulate ribosomal DNA replication timing via MCM helicase positioning and nucleosome occupancy.

The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well-known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). We have previously reported that in the absence of SIR2, a derepressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both, activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

Assessing contributions of DNA sequences at the 3’ end of a yeast gene on yFACT, RNA polymerase II, and nucleosome occupancy

ObjectiveIn past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3’ end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3’ end of genes in promoting yFACT dissociation upon transcription termination.ResultsWe generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3’ end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene’s 3’ end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3’ ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture.

GuidedNOMe-seq quantifies chromatin states at single allele resolution for hundreds of custom regions in parallel

Since the introduction of next generation sequencing technologies, the field of epigenomics has evolved rapidly. However, most commonly used assays are enrichment-based methods and thus only semi-quantitative. Nucleosome occupancy and methylome sequencing (NOMe-seq) allows for quantitative inference of chromatin states with single locus resolution, but this requires high sequencing depth and is therefore prohibitively expensive to routinely apply to organisms with large genomes. To overcome this limitation, we introduce guidedNOMe-seq, where we combine NOMe profiling with large scale sgRNA synthesis and Cas9-mediated region-of-interest (ROI) liberation. To facilitate quantitative comparisons between multiple samples, we additionally develop an R package to standardize differential analysis of any type of NOMe-seq data. We extensively benchmark guidedNOMe-seq in a proof-of-concept study, dissecting the interplay of ChAHP and CTCF on chromatin. In summary we present a cost-effective, scalable, and customizable target enrichment extension to the existing NOMe-seq protocol allowing genome-scale quantification of nucleosome occupancy and transcription factor binding at single allele resolution.

Remodeling of perturbed chromatin can initiate de novo transcriptional and post-transcriptional silencing

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.

PICKLE and HISTONE DEACETYLASE6 coordinately regulate genes and transposable elements in Arabidopsis.

Chromatin dynamics play essential roles in transcriptional regulation. The chromodomain helicase DNA-binding domain 3 chromatin remodeler PICKLE (PKL) and HISTONE DEACETYLASE6 (HDA6) are required for transcriptional gene silencing, but their coordinated function in gene repression requires further study. Through a genetic suppressor screen, we found that a point mutation at PKL could partially restore the developmental defects of a weak Polycomb repressive complex 1 (PRC1) mutant (ring1a-2 ring1b-3), in which RING1A expression is suppressed by a T-DNA insertion at the promoter. Compared to ring1a-2 ring1b-3, the expression of RING1A is increased, nucleosome occupancy is reduced, and the histone 3 lysine 9 acetylation (H3K9ac) level is increased at the RING1A locus in the pkl ring1a-2 ring1b-3 triple mutant. HDA6 interacts with PKL and represses RING1A expression similarly to PKL genetically and molecularly in the ring1a-2 ring1b-3 background. Furthermore, we show that PKL and HDA6 suppress the expression of a set of genes and transposable elements (TEs) by increasing nucleosome density and reducing H3K9ac. Genome-wide analysis indicated they possibly coordinately maintain DNA methylation as well. Our findings suggest that PKL and HDA6 function together to reduce H3K9ac and increase nucleosome occupancy, thereby facilitating gene/TE regulation in Arabidopsis (Arabidopsis thaliana).

P-523 Single molecule chromatin footprinting reveals only low occurrence of nucleosomes at multiple loci throughout chromatin of human and mouse spermatozoa

Abstract Study question What fraction of spermatozoa from mouse and human semen contain nucleosomes at specific loci? What chromatin features affect nucleosome retention in spermatozoa? Summary answer On average, 1-2% mouse spermatozoa and 5-9% human spermatozoa contain nucleosomes at sampled genomic loci, with moderately higher frequencies at unmethylated regions of DNA. What is known already Nucleosomes constitute the basic packaging and gene regulatory unit of DNA in eukaryotic cells. During final stages of spermatogenesis in mammals, however, most nucleosomes are replaced by protamines. Previous global chromatin immunoprecipitation-sequencing studies reported the presence of nucleosomes at gene regulatory and other genomic regions. Such nucleosomes were proposed to convey intergenerational inheritance of epigenetic information. Additional studies revealed that infertile men with decreased success of assisted reproductive therapy have protamine deficiency or displayed more widely distributed nucleosomes in their sperm genome. Direct links between altered levels of nuclear proteins and fertilizing potential of sperm have, however, not been established. Study design, size, duration We conducted a case study on nucleosome retention levels in sperm from four selected fertile human samples. This study was complemented in mice, in which we characterized the dynamic changes in nucleosome frequencies at genomic loci during differentiation of haploid spermatids into spermatozoa, and their responses in mutant spermatozoa lacking DNA methylation at certain loci. Participants/materials, setting, methods We used the single-molecule sequencing technique called Nucleosome Occupancy and Methylome Sequencing to quantify nucleosome occupancy rates at selected loci in spermatozoa from ejaculated semen of four fertile semen donors. We also profiled round spermatids, early and late elongating spermatids and spermatozoa of C57BL/6J wild-type mice as well as spermatozoa of mice conditionally deficient for the methyltransferases Dnmt3a and Dnmt3b, lacking DNA methylation at certain genomic regions. Main results and the role of chance To our knowledge, this is the first study to quantify nucleosome occupancy in mouse male gametes and human spermatozoa at single-molecule resolution. In mice, we observed that chromatin remodelling during spermatid elongation resulted in a progressive loss of nucleosomes and their positioning, a transient gain in global chromatin accessibility which was succeeded by increasing chromatin inaccessibility and nuclear compaction. In both human and mouse sperm, we profiled nucleosome occupancy levels at different genomic loci and measured only low percentages, ranging from ∼1-2% in mouse and 5-9% in human. In both organisms we observed a near two-fold higher nucleosome occupancy at loci being unmethylated at their DNA over loci being highly methylated. Mice partially deficient in genomic DNA methylation manifested a similar fold increase in nucleosome occupancy in absence of DNA methylation. These data reveal a moderate modulatory role for DNA methylation established in spermatogonial stages in regulating nucleosome eviction during spermiogenesis. The overall low nucleosome occupancy frequencies in mammalian spermatozoa suggest a stochastic or random rather than a programmed role of nucleosomes in regulating paternal gene expression during ensuing embryonic development. Limitations, reasons for caution Amplicon-based sequencing provides deep sequencing coverage for selected genomic loci. We lack, however, information on the distribution of nucleosomes among loci within the genome of single spermatozoa. In human, we studied a limited number of human donors. Wider implications of the findings This study resolves a long-standing question on the frequency and local distribution of nucleosomes in murine and human spermatozoa. The variability in nucleosome occupancy between spermatozoa within and between samples of human individuals inspires further investigations into the relationship between chromatin states and quality of individual spermatozoa for embryonic development. Trial registration number SNCTP000003654

MacroH2A1 drives nucleosome dephasing and genome instability in histone humanized yeast

In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

Characterization of the SWI/SNF complex and nucleosome organization in sorghum.

The switch defective/sucrose non-fermentable (SWI/SNF) multisubunit complex plays an important role in the regulation of gene expression by remodeling chromatin structure. Three SWI/SNF complexes have been identified in Arabidopsis including BAS, SAS, and MAS. Many subunits of these complexes are involved in controlling plant development and stress response. However, the function of these complexes has hardly been studied in other plant species. In this study, we identified the subunits of the SWI/SNF complex in sorghum and analyzed their evolutionary relationships in six grass species. The grass species conserved all the subunits as in Arabidopsis, but gene duplication occurred diversely in different species. Expression pattern analysis in sorghum (Sorghum bicolor) showed that most of the subunit-encoding genes were expressed constitutively, although the expression level was different. Transactivation assays revealed that SbAN3, SbGIF3, and SbSWI3B possessed transactivation activity, which suggests that they may interact with the pre-initiation complex (PIC) to activate transcription. We chose 12 subunits in sorghum to investigate their interaction relationship by yeast two-hybrid assay. We found that these subunits displayed distinct interaction patterns compared to their homologs in Arabidopsis and rice. This suggests that different SWI/SNF complexes may be formed in sorghum to perform chromatin remodeling functions. Through the integrated analysis of MNase-seq and RNA-seq data, we uncovered a positive relationship between gene expression levels and nucleosome phasing. Furthermore, we found differential global nucleosome enrichments between leaves and roots, as well as in response to PEG treatment, suggesting that dynamics of nucleosome occupancy, which is probably mediated by the SWI/SNF complex, may play important roles in sorghum development and stress response.

A nucleosome switch primes Hepatitis B Virus infection.

Chronic hepatitis B virus (HBV) infection is an incurable global health threat responsible for causing liver disease and hepatocellular carcinoma. During the genesis of infection, HBV establishes an independent minichromosome consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. The viral X gene must be expressed immediately upon infection to induce degradation of the host silencing factor, Smc5/6. However, the relationship between cccDNA chromatinization and X gene transcription remains poorly understood. Establishing a reconstituted viral minichromosome platform, we found that nucleosome occupancy in cccDNA drives X transcription. We corroborated these findings in cells and further showed that the chromatin destabilizing molecule CBL137 inhibits X transcription and HBV infection in hepatocytes. Our results shed light on a long-standing paradox and represent a potential new therapeutic avenue for the treatment of chronic HBV infection.

The PCNA-Pol δ complex couples lagging strand DNA synthesis to parental histone transfer for epigenetic inheritance.

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.

Circadian control of histone turnover during cardiac development and growth

During postnatal cardiac hypertrophy, cardiomyocytes undergo mitotic exit, relying on DNA replication-independent mechanisms of histone turnover to maintain chromatin organization and gene transcription. In other tissues, circadian oscillations in nucleosome occupancy influence clock-controlled gene expression, suggesting a role for the circadian clock in temporal control of histone turnover and coordinated cardiomyocyte gene expression. To elucidate roles for the master circadian transcription factor, Bmal1, in histone turnover, chromatin organization, and myocyte-specific gene expression and cell growth in the neonatal period. Bmal1 knockdown in neonatal rat ventricular myocytes (NRVM) decreased myocyte size, total cellular protein synthesis, and transcription of the fetal hypertrophic gene Nppb following treatment with serum or the α-adrenergic agonist phenylephrine (PE). Depletion of Bmal1 decreased expression of clock-controlled genes Per2 and Tcap, as well as Sik1, a Bmal1 target upregulated in adult versus embryonic hearts. Bmal1 knockdown impaired Per2 and Sik1 promoter accessibility as measured by MNase-qPCR and impaired histone turnover as measured by metabolic labeling of acid-soluble chromatin fractions. Sik1 knockdown in turn decreased myocyte size, while simultaneously inhibiting Nppb transcription and activating Per2 transcription. Linking these changes to chromatin remodeling, depletion of the replication-independent histone variant H3.3a inhibited myocyte hypertrophy and prevented PE-induced changes in clock-controlled gene transcription. Bmal1 is required for neonatal myocyte growth, replication-independent histone turnover, and chromatin organization at the Sik1 promoter. Sik1 represents a novel clock-controlled gene that coordinates myocyte growth with hypertrophic and clock-controlled gene transcription. Replication-independent histone turnover is required for transcriptional remodeling of clock-controlled genes in cardiac myocytes in response to growth stimuli.

Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

Genome-wide nucleosome and transcription factor responses to genetic perturbations reveal chromatin-mediated mechanisms of transcriptional regulation.

Epigenetic mechanisms contribute to gene regulation by altering chromatin accessibility through changes in transcription factor (TF) and nucleosome occupancy throughout the genome. Despite numerous studies focusing on changes in gene expression, the intricate chromatin-mediated regulatory code remains largely unexplored on a comprehensive scale. We address this by employing a factor-agnostic, reverse-genetics approach that uses MNase-seq to capture genome-wide TF and nucleosome occupancies in response to the individual deletion of 201 transcriptional regulators in Saccharomyces cerevisiae, thereby assaying nearly one million mutant-gene interactions. We develop a principled approach to identify and quantify chromatin changes genome-wide, observing differences in TF and nucleosome occupancy that recapitulate well-established pathways identified by gene expression data. We also discover distinct chromatin signatures associated with the up- and downregulation of genes, and use these signatures to reveal regulatory mechanisms previously unexplored in expression-based studies. Finally, we demonstrate that chromatin features are predictive of transcriptional activity and leverage these features to reconstruct chromatin-based transcriptional regulatory networks. Overall, these results illustrate the power of an approach combining genetic perturbation with high-resolution epigenomic profiling; the latter enables a close examination of the interplay between TFs and nucleosomes genome-wide, providing a deeper, more mechanistic understanding of the complex relationship between chromatin organization and transcription.

General regulatory factors exert differential effects on nucleosome sliding activity of the ISW1a complex

BackgroundChromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown.ResultsConsidering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome.ConclusionsOur findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.

Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.

Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (dcp2Δ), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2Δ, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2∆, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.

Study on chromatin regulation patterns of expression vectors in the PhiC31 integration site

ABSTRACT The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.