strand-specific-analysis-proteins-at-replicating-dna-strands

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

University of Minnesota

Liquid phase condensation directs nucleosome epigenetic modifications.

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in  mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in  and  cells defective in replication checkpoint but, surprisingly, not in  cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in  mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast.

Chromatin structure contains critical epigenetic information in various forms, such as histone post-translational modifications (PTMs). The deposition of certain histone PTMs can remodel the chromatin structure, resulting in gene expression alteration. The epigenetic information carried by histone PTMs could be inherited by daughter cells to maintain the gene expression status. Recently, studies revealed that several conserved replisome proteins regulate the recycling of parental histones carrying epigenetic information in . Hence, the proper recycling and deposition of parental histones onto newly synthesized DNA strands is presumed to be essential for epigenetic inheritance. Here, we first reviewed the fundamental mechanisms of epigenetic modification establishment and maintenance discovered within fungal models. Next, we discussed the functions of parental histone chaperones and the potential impacts of the parental histone recycling process on heterochromatin-mediated transcriptional silencing inheritance. Subsequently, we summarized novel synthetic biology approaches developed to analyze individual epigenetic components during epigenetic inheritance in fungal and mammalian systems. These newly emerged research paradigms enable us to dissect epigenetic systems in a bottom-up manner. Furthermore, we highlighted the approaches developed in this emerging field and discussed the potential applications of these engineered regulators to building synthetic epigenetic systems.

Advances in Approaches to Study Chromatin-Mediated Epigenetic Memory.

Eukaryotic Macrotransposons (MTns) can be formed by 2 nearby elements flanking a segment of host DNA. The maize Ac transposon can form Ac::MTns, but little is known about Ac::MTn transposition activities. Here, we studied 3 Ac::MTns at the maize p1 locus, each of which is composed of a segment of maize p1 genomic DNA (up to 15 kb) bounded by a fractured Ac element (fAc, 2039 bp), and a full-length Ac element in direct orientation. The resulting Ac::MTns are of 16, 16.5, and 22 kb total length. From these 3 Ac::MTns, we identified 10 independent cases of macrotransposition, and observed similar features of transposition between Ac::MTn and standard Ac/Ds, including characteristic excision footprints and insertion target site duplications. Nine out of the 10 Ac::MTn reinsertion targets were genetically linked to the donor sites, another similarity with Ac/Ds standard transposition. We also identified a MTn-like structure in the maize B73 reference genome and 5 NAM founder lines. The MTn in diverse lines is flanked by target site duplications, confirming the historic occurrence of MTn transposition during genome evolution. Our results show that Ac::MTns are capable of mobilizing segments of DNA long enough to include a typical full-length plant gene and in theory could erode gene colinearity in syntenic regions during plant genome evolution.

Excision and reinsertion of Ac macrotransposons in maize.

During DNA replication, parental H3-H4 marked by H3K4me3 are transferred almost equally onto leading and lagging strands of DNA replication forks. Mutations in replicative helicase subunit, Mcm2 (Mcm2-3A), and leading strand DNA polymerase subunit, Dpb3 (∆), result in asymmetric distributions of H3K4me3 at replicating DNA strands immediately following DNA replication. Here, we show that  and ∆ mutant cells markedly reduce the asymmetric distribution of H3K4me3 during cell cycle progression before mitosis. Furthermore, the restoration of a more symmetric distribution of H3K4me3 at replicating DNA strands in these mutant cells is driven by methylating nucleosomes without H3K4me3 by the H3K4 methyltransferase complex, COMPASS. Last, both gene transcription machinery and the binding of parental H3K4me3 by Spp1 subunit of the COMPASS complex help recruit the enzyme to chromatin for the restoration of the H3K4me3-marked state following DNA replication, shedding light on inheritance of this mark following DNA replication.

H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication.

Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes by regulating the activity of their enhancers. BTKi treatment reduces enrichment of enhancer marks (H3K4me1 and H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. We also find that enhancer-regulated APOBEC3 expression contributes to replication stress in malignant B cells. In total we demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in an NFATc1-dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.

B cell receptor signaling drives APOBEC3 expression via direct enhancer regulation in chronic lymphocytic leukemia B cells.

Faithful inheritance of parental histones is essential to maintain epigenetic information and cellular identity during cell division. Parental histones are evenly deposited onto the replicating DNA of sister chromatids in a process dependent on the MCM2 subunit of DNA helicase. However, the impact of aberrant parental histone partition on human disease such as cancer is largely unknown. In this study, we construct a model of impaired histone inheritance by introducing MCM2-2A mutation (defective in parental histone binding) in MCF-7 breast cancer cells. The resulting impaired histone inheritance reprograms the histone modification landscapes of progeny cells, especially the repressive histone mark H3K27me3. Lower H3K27me3 levels derepress the expression of genes associated with development, cell proliferation, and epithelial to mesenchymal transition. These epigenetic changes confer fitness advantages to some newly emerged subclones and consequently promote tumor growth and metastasis after orthotopic implantation. In summary, our results indicate that impaired inheritance of parental histones can drive tumor progression.

Impaired histone inheritance promotes tumor progression.

Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice.

Parental histones, the carriers of posttranslational modifications, are deposited evenly onto the replicating DNA of sister chromatids in a process dependent on the Mcm2 subunit of DNA helicase and the Pole3 subunit of leading-strand DNA polymerase. The biological significance of parental histone propagation remains unclear. Here we show that Mcm2-mutated or Pole3-deleted mouse embryonic stem cells (ESCs) display aberrant histone landscapes and impaired neural differentiation. Mutation of the Mcm2 histone-binding domain causes defects in pre-implantation development and embryonic lethality. ESCs with biased parental histone transfer exhibit increased epigenetic heterogeneity, showing altered histone variant H3.3 and H3K27me3 patterning at genomic sites regulating differentiation genes. Our results indicate that the lagging strand pattern of H3.3 leads to the redistribution of H3K27me3 in Mcm2-2A ESCs. We demonstrate that symmetric parental histone deposition to sister chromatids contributes to cellular differentiation and development.

Symmetric inheritance of parental histones contributes to safeguarding the fate of mouse embryonic stem cells during differentiation.

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells.

Defective transfer of parental histone decreases frequency of homologous recombination by increasing free histone pools in budding yeast.

Asymmetric inheritance of parental histones leads to differentiation defects in mouse embryonic stem cells.

Chromatin replication is intricately intertwined with the recycling of parental histones to the newly duplicated DNA strands for faithful genetic and epigenetic inheritance. The transfer of parental histones occurs through two distinct pathways: leading strand deposition, mediated by the DNA polymerase ε subunits Dpb3/Dpb4, and lagging strand deposition, facilitated by the MCM helicase subunit Mcm2. However, the mechanism of the facilitation of Mcm2 transferring parental histones to the lagging strand while moving along the leading strand remains unclear. Here, we show that the deletion of Pol32, a nonessential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the leading strand during replication. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted through the mutation of the histone H3-H4 binding domain within Mcm2. Our findings identify the DNA polymerase δ subunit Pol32 as a critical histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

DNA polymerase delta governs parental histone transfer to DNA replication lagging strand.

Human hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication-coupled epigenetic inheritance process, and chromatin stability.

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast.

Recycling histone proteins from parental chromatin, a process known as parental histone transfer, is an important component in chromosome replication and is essential for epigenetic inheritance. We review recent advances in our understanding of the recycling mechanism of parental histone H3-H4 tetramers (parH3:H4tet), emphasizing the pivotal role of the DNA replisome. In particular, we highlight the function of the MCM2-7 helicase subunit Mcm2 as a histone H3-H4 tetramer chaperone. Disruption of this histone chaperone's functions affects mouse embryonic stem cell differentiation and can lead to embryonic lethality in mice, underscoring the crucial role of the replisome in maintaining epigenomic stability.

The Role of the MCM2-7 Helicase Subunit MCM2 in Epigenetic Inheritance.

Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells. However, the functional significance of ecDNA and the molecular components involved in its replication and maintenance remain largely unknown. Here, using CRISPR-C technology, we generated ecDNA-carrying (ecDNA+) cell models. By leveraging these models alongside other well-established systems, we demonstrated that ecDNA can replicate and be maintained in ecDNA+ cells. The replication of ecDNA activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. Topoisomerases, such as TOP1 and TOP2B, play a role in ecDNA replication-induced DNA double-strand breaks (DSBs). A subset of these elevated DSBs persists into the mitotic phase and is primarily repaired by the alternative non-homologous end joining (alt-NHEJ) pathway, which involves POLθ and LIG3. Correspondingly, ecDNA maintenance requires DDR, and inhibiting DDR impairs the circularization of ecDNA. In summary, we demonstrate reciprocal interactions between ecDNA maintenance and DDR, providing new insights into the detection and treatment of ecDNA+ tumors.

Article	Year
Liquid phase condensation directs nucleosome epigenetic modifications. Signal transduction and targeted therapy\| PubMed ID: 32376821	2020
A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34531325	2021
Advances in Approaches to Study Chromatin-Mediated Epigenetic Memory. ACS synthetic biology\| PubMed ID: 34965084	2022
Excision and reinsertion of Ac macrotransposons in maize. Genetics\| PubMed ID: 35471241	2022
H3K4me3 recognition by the COMPASS complex facilitates the restoration of this histone mark following DNA replication. Science advances\| PubMed ID: 35544640	2022
B cell receptor signaling drives APOBEC3 expression via direct enhancer regulation in chronic lymphocytic leukemia B cells. Blood cancer journal\| PubMed ID: 35778390	2022
Impaired histone inheritance promotes tumor progression. Nature communications\| PubMed ID: 37301892	2023
Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice. Gastroenterology\| PubMed ID: 37597632	2023
Symmetric inheritance of parental histones contributes to safeguarding the fate of mouse embryonic stem cells during differentiation. Nature genetics\| PubMed ID: 37666989	2023
Defective transfer of parental histone decreases frequency of homologous recombination by increasing free histone pools in budding yeast. Nucleic acids research\| PubMed ID: 38554108	2024
Asymmetric inheritance of parental histones leads to differentiation defects in mouse embryonic stem cells. The innovation life\| PubMed ID: 38601056	2024
DNA polymerase delta governs parental histone transfer to DNA replication lagging strand. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 38713623	2024
The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast. Epigenetics & chromatin\| PubMed ID: 39192292	2024
The Role of the MCM2-7 Helicase Subunit MCM2 in Epigenetic Inheritance. Biology\| PubMed ID: 39194510	2024
Extrachromosomal DNA replication and maintenance couple with DNA damage pathway in tumors. Cell\| PubMed ID: 40300601	2025

Chuanhe Yu

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