Nucleoplasmic Extracts Research Articles

Single-molecule study reveals Hmo1, not Hho1, promotes chromatin assembly in budding yeast.

Linker histone H1 plays a crucial role in various biological processes, including nucleosome stabilization, high-order chromatin structure organization, gene expression, and epigenetic regulation in eukaryotic cells. Unlike higher eukaryotes, little about the linker histone in Saccharomyces cerevisiae is known. Hho1 and Hmo1 are two long-standing controversial histone H1 candidates in budding yeast. In this study, we directly observed at the single-molecule level that Hmo1, but not Hho1, is involved in chromatin assembly in the yeast nucleoplasmic extracts (YNPE), which can replicate the physiological condition of the yeast nucleus. The presence of Hmo1 facilitates the assembly of nucleosomes on DNA in YNPE, as revealed by single-molecule force spectroscopy. Further single-molecule analysis showed that the lysine-rich C-terminal domain (CTD) of Hmo1 is essential for the function of chromatin compaction, while the second globular domain at the C-terminus of Hho1 impairs its ability. In addition, Hmo1, but not Hho1, forms condensates with double-stranded DNA via reversible phase separation. The phosphorylation fluctuation of Hmo1 coincides with metazoan H1 during the cell cycle. Our data suggest that Hmo1, but not Hho1, possesses some functionality similar to that of linker histone in Saccharomyces cerevisiae, even though some properties of Hmo1 differ from those of a canonical linker histone H1. Our study provides clues for the linker histone H1 in budding yeast and provides insights into the evolution and diversity of histone H1 across eukaryotes. IMPORTANCE There has been a long-standing debate regarding the identity of linker histone H1 in budding yeast. To address this issue, we utilized YNPE, which accurately replicate the physiological conditions in yeast nuclei, in combination with total internal reflection fluorescence microscopy and magnetic tweezers. Our findings demonstrated that Hmo1, rather than Hho1, is responsible for chromatin assembly in budding yeast. Additionally, we found that Hmo1 shares certain characteristics with histone H1, including phase separation and phosphorylation fluctuations throughout the cell cycle. Furthermore, we discovered that the lysine-rich domain of Hho1 is buried by its second globular domain at the C-terminus, resulting in the loss of function that is similar to histone H1. Our study provides compelling evidence to suggest that Hmo1 shares linker histone H1 function in budding yeast and contributes to our understanding of the evolution of linker histone H1 across eukaryotes.

Transcription suppression is mediated by the HDAC1–Sin3 complex in Xenopus nucleoplasmic extract

Modification of histones provides a dynamic mechanism to regulate chromatin structure and access to DNA. Histone acetylation, in particular, plays a prominent role in controlling the interaction between DNA, histones, and other chromatin-associated proteins. Defects in histone acetylation patterns interfere with normal gene expression and underlie a wide range of human diseases. Here, we utilize Xenopus egg extracts to investigate how changes in histone acetylation influence transcription of a defined gene construct. We show that inhibition of histone deacetylase 1 and 2 (HDAC1/2) specifically counteracts transcription suppression by preventing chromatin compaction and deacetylation of histone residues H4K5 and H4K8. Acetylation of these sites supports binding of the chromatin reader and transcription regulator BRD4. We also identify HDAC1 as the primary driver of transcription suppression and show that this activity is mediated through the Sin3 histone deacetylase complex. These findings highlight functional differences between HDAC1 and HDAC2, which are often considered to be functionally redundant, and provide additional molecular context for their activity.

Identification of Pri-miRNA Stem-Loop Interacting Proteins in Plants Using a Modified Version of the Csy4 CRISPR Endonuclease.

Regulation at the RNA level by RNA-binding proteins (RBPs) and microRNAs (miRNAs) is key to coordinating eukaryotic gene expression. In plants, the importance of miRNAs is highlighted by severe developmental defects in mutants impaired in miRNA biogenesis. MiRNAs are processed from long primary-microRNAs (pri-miRNAs) with internal stem-loop structures by endonucleolytic cleavage. The highly structured stem-loops constitute the basis for the extensive regulation of miRNA biogenesis through interaction with RBPs. However, trans-acting regulators of the biogenesis of specific miRNAs are largely unknown in plants. Therefore, we exploit an RNA-centric approach based on modified versions of the conditional CRISPR nuclease Csy4* to pull down interactors of the Arabidopsis pri-miR398b stem-loop (pri-miR398b-SL) in vitro. We designed three epitope-tagged versions of the inactive Csy4* for the immobilization of the protein together with the pri-miR398b-SL bait on high affinity matrices. After incubation with nucleoplasmic extracts from Arabidopsis and extensive washing, pri-miR398b-SL, along with its specifically bound proteins, were released by re-activating the cleavage activity of the Csy4* upon the addition of imidazole. Co-purified proteins were identified via quantitative mass spectrometry and data sets were compared. In total, we identified more than 400 different proteins, of which 180 are co-purified in at least two out of three independent Csy4*-based RNA pulldowns. Among those, the glycine-rich RNA-binding protein AtRZ-1a was identified in all pulldowns. To analyze the role of AtRZ-1a in miRNA biogenesis, we determined the miR398 expression level in the atrz-1a mutant. Indeed, the absence of AtRZ-1a caused a decrease in the steady-state level of mature miR398 with a concomitant reduction in pri-miR398b levels. Overall, we show that our modified Csy4*-based RNA pulldown strategy is suitable to identify new trans-acting regulators of miRNA biogenesis and provides new insights into the post-transcriptional regulation of miRNA processing by plant RBPs.

In Vitro FRET‐Based Helicase Activity Assay as a Drug Screening Tool

The Cdc45‐Mcm2‐7‐GINS (CMG) helicase is a protein complex that unwinds double‐stranded DNA into single strands during DNA replication at the S‐phase. Previous research has pointed to the CMG helicase as a potential target for cancer treatment. Indeed, the CMG helicase activity is an essential and vulnerable component of the cell cycle machinery of cancerous cells. However, the development of anti‐cancer helicase targeting molecules requires a robust and quantifiable helicase assay.Here we report a novel CMG helicase activity assay that utilizes FAM‐BlackFQ dual‐labeled DNA oligonucleotides as a substrate. The underlying principle of the Fluorescence Resonance Energy Transfer (FRET) allows for a quick, reliable, real‐time measurement of helicase activity in vitro. As a source of active CMG helicases, we prepared a nucleoplasmic extract (NPE) derived from the cell‐free system of Xenopus egg extracts, a canonical system widely used to study vertebrate DNA replication in vitro. This NPE is a rich source of the Xenopus CMG helicase, which is highly analogous to its human counterpart.In this report, we present optimization of our novel assay. Remediation of potential contaminations with endogenous DNases is addressed. We utilized our helicase assay to study inhibition of the CMG helicase by therapeutically relevant peptides. We also extended its utility by screening natural compounds of the NCI/DTP library N5.

BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract.

The tumor suppressor BRCA1 is considered a master regulator of genome integrity. Although widely recognized for its DNA repair functions, BRCA1 has also been implicated in various mechanisms of chromatin remodeling and transcription regulation. However, the precise role that BRCA1 plays in these processes has been difficult to establish due to the widespread consequences of its cellular dysfunction. Here, we use nucleoplasmic extract derived from the eggs of Xenopus laevis to investigate the role of BRCA1 in a cell-free transcription system. We report that BRCA1-BARD1 suppresses transcription initiation independent of DNA damage signaling and its established role in histone H2A ubiquitination. BRCA1-BARD1 acts through a histone intermediate, altering acetylation of histone H4K8 and recruitment of the chromatin reader and oncogene regulator BRD4. Together, these results establish a functional relationship between an established (BRCA1) and emerging (BRD4) regulator of genome integrity.

Cell-free transcription in Xenopus egg extract

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that Xenopus nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts.

Extracts for Analysis of DNA Replication in a Nucleus-Free System.

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.

Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts

In eukaryotic cells, the smallest subunit of chromatin is the nucleosome, which consists of a segment of DNA wound on histone protein cores. Despite many years of effort, the process of nucleosome assembly and disassembly is still not very clear. Here, we present a convenient method to investigate the process of nucleosome assembly at the single molecule level. We invented a novel system derived from the yeast nucleoplasmic extracts (YNPE), and demonstrated that the YNPE supports the nucleosome assembly under physiological condition. By combining the total internal reflection fluorescence microscopy with microfluidic flow-cell technique, the dynamic process of nucleosome assembly in YNPE was visualized at single-molecule level. Our system provides a novel in vitro single-molecule tool to investigate the dynamics of nucleosome assembly under physiological conditions.

Preparation of Ribonucleoprotein-Enriched Nucleoplasmic Extracts from Drosophila Cells

Here we describe how to prepare nucleoplasmic extracts from suspension cultures of Drosophila S2 (Schneider Line 2) cells. Harvested cells are washed in phosphate-buffered saline supplemented with MgCl2, resuspended in hypotonic buffer, and homogenized. Nuclei are isolated by centrifugation and then sonicated. The nuclear sonicate is placed on a 30% sucrose cushion and sedimented. The soluble nuclear ribonucleoprotein (RNP) complexes remain in the supernatant and the nuclear membrane fragments and chromatin pellet through the sucrose cushion.

Replication protein A promotes 5′→3′ end processing during homology-dependent DNA double-strand break repair

Replication protein A (RPA), the eukaryotic single-strand deoxyribonucleic acid (DNA [ss-DNA])-binding protein, is involved in DNA replication, nucleotide damage repair, mismatch repair, and DNA damage checkpoint response, but its function in DNA double-strand break (DSB) repair is poorly understood. We investigated the function of RPA in homology-dependent DSB repair using Xenopus laevis nucleoplasmic extracts as a model system. We found that RPA is required for single-strand annealing, one of the homology-dependent DSB repair pathways. Furthermore, RPA promotes the generation of 3' single-strand tails (ss-tails) by stimulating both the Xenopus Werner syndrome protein (xWRN)-mediated unwinding of DNA ends and the subsequent Xenopus DNA2 (xDNA2)-mediated degradation of the 5' ss-tail. Purified xWRN, xDNA2, and RPA are sufficient to carry out the 5'-strand resection of DNA that carries a 3' ss-tail. These results provide strong biochemical evidence to link RPA to a specific DSB repair pathway and reveal a novel function of RPA in the generation of 3' ss-DNA for homology-dependent DSB repair.

Inhibition of nucleosome assembly by the mismatch repair system

The mismatch repair (MMR) system post‐replicatively corrects replication errors such as base‐base mismatches. The MMR reaction begins with recognition of the errors by the MutS complex. MutS and other factors then identify the erroneous newly synthesized strand. The nature of strand discrimination signals in eukaryotes remains elusive, but a gap or 3′‐end of DNA can work as the signal in vitro. Concurrent with the MMR reaction, nucleosomes re‐assemble on the replicated DNA strands. Nucleosome assembly might impede the MMR reaction, for example by obscuring strand discrimination signals. However, how the MMR system deals with nucleosome assembly remains highly unclear.Using Xenopus egg extracts as a vertebrate model system, we investigated functional interaction between the MMR reaction and nucleosome assembly. In the nucleoplasmic extracts of Xenopus eggs, nucleosomes rapidly assemble on naked DNA. Surprisingly, nucleosome assembly was severely inhibited when a base‐base mismatch was present on the DNA molecule. The MutS complex specifically bound to the mismatch‐containing DNA, and this complex was essential for both repair of the mismatch and inhibition of nucleosome assembly. These results suggest that nucleosome assembly at replication error loci is temporally inhibited by a MutS‐dependent mechanism during the MMR reaction. We propose that this mechanism is important for eukaryotic MMR in the context of chromatin.

Analysis of histones and chromatin in Xenopus laevis egg and oocyte extracts

Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract (“NPE”); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system.

In new company: U1 snRNA associates with TAF15

In new company: U1 snRNA associates with TAF15

Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.

Analyzing the ATR-mediated checkpoint using Xenopus egg extracts

Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.

Mechanistic Analysis of a DNA End Processing Pathway Mediated by the Xenopus Werner Syndrome Protein

The first step of homology-dependent repair of DNA double-strand breaks is the strand-specific processing of DNA ends to generate 3' single-strand tails. Despite its importance, the molecular mechanism underlying end processing is poorly understood in eukaryotic cells. We have taken a biochemical approach to investigate DNA end processing in nucleoplasmic extracts derived from the unfertilized eggs of Xenopus laevis. We found that double-strand DNA ends are specifically degraded in the 5' --> 3' direction in this system. The reaction consists of two steps: an ATP-dependent unwinding of double-strand ends and an ATP-independent 5' --> 3' degradation of single-strand tails. We also found that the Xenopus Werner syndrome protein, a member of the RecQ helicase family, plays an important role in DNA end processing. Mechanistically, Xenopus Werner syndrome protein (xWRN) is required for the unwinding of DNA ends but not for the degradation of single-strand tails. The xWRN-mediated end processing is remarkably similar to the end processing that has been proposed for the Escherichia coli RecQ helicase and RecJ single-strand nuclease, suggesting that this mechanism might be conserved in prokaryotes and eukaryotes.

Proliferating Cell Nuclear Antigen Recruits Cyclin-dependent Kinase Inhibitor Xic1 to DNA and Couples Its Proteolysis to DNA Polymerase Switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.

DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activities in vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) from Xenopus laevis eggs and six purified polyclonal antibodies generated against each of the Xenopus Mcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2-7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21(cip1) specifically blocks origin firing but does not prevent helicase action. When p21(cip1) is added, followed by the non-hydrolyzable analog ATPgammaS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

Connecting the Checkpoint with Repair

DNA damage can occur through several mechanisms. There can be breakage of the strands or chemical alterations to the DNA, such as alkylation. Double-stranded breaks and alkylation activate different checkpoint pathways, allowing the cell time to repair the damage. Two groups studied the DNA repair and checkpoint signaling activated by alkylation damage in response to methyl methanesulfonate (MMS). Stokes et al. analyzed the requirement for DNA replication during activation of the alkylation checkpoint pathway, using in vitro systems based on Xenopus oocyte extracts or nucleoplasmic extracts. The addition of MMS-treated sperm chromatin to the cycling egg extracts activated the checkpoint kinase ATR, promoting the phosphorylation of one of the ATR substrates Chk-1, and delayed progression through mitosis when compared to egg extracts to which normal sperm chromatin had been added. Activation of ATR and the delayed progression through mitosis were blocked if DNA replication or entry into S phase was inhibited, suggesting that DNA replication may be required for activation of the checkpoint responsible for detection of alkylated DNA. Furthermore, Rad17, a protein that is part of a complex involved in loading the PCNA protein onto replicating DNA, was found to associate specifically with the alkylated DNA and this association required DNA replication. The second group, Hoege et al. , demonstrated that DNA repair involves the PCNA protein and its modification by ubiquitin. In Saccharomyces cerevisiae , PCNA was modified by both ubiquitin and the ubiquitin-related protein SUMO on the same lysine residue. Sumoylation is stimulated during S phase under normal conditions with undamaged DNA. Treatment of the yeast with MMS induced the ubiquitination of PCNA, and this was blocked if genes of the RAD6 repair pathway were mutated. RAD6 is a ubiquitin-conjugating enzyme, which works together with another ubiquitin-conjugating complex, UBC13-MMS2, and the DNA binding RING finger ubiquitin ligases RAD18 and RAD5 in postreplication DNA repair. Genetic interactions occurred between PCNA and these RAD6 pathway genes, altering the ability of the yeast to survive MMS treatment. It will be interesting to see if the Rad17 interaction noted by Stokes et al. and the PCNA modifications reported by Hoege et al. converge on the selective recruitment of differently modified versions of PCNA onto the damaged DNA. M. P. Stokes, R. Van Hatten, H. D. Lindsay, W. M. Michael, DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts. J. Cell Biol. 158 , 863-872 (2002). [Abstract] [Full Text] C. Hoege, B. Pfander, G.-L. Moldovan, G. Pyrowolakis, S. Jentsch, RAD6 -dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419 , 135-141 (2002). [Online Journal]

Evidence for Sequential Action of cdc7 and cdk2 Protein Kinases during Initiation of DNA Replication in Xenopus Egg Extracts

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.