Native nucleosome-positioning elements as alternatives to the 601 sequence for nucleosome repositioning studies.

Nucleosome repositioning is essential for establishing nucleosome-depleted regions to initiate transcription. This process has been extensively studied using structural, biochemical, and single-molecule approaches, which require homogeneously positioned nucleosomes. This is often achieved using the Widom 601 sequence, a highly efficient nucleosome-positioning element (NPE) selected for its unusually strong binding to the H3-H4 histone tetramer.

GAGA zinc finger transcription factor searches chromatin by 1D-3D facilitated diffusion.

The search for target sites on chromatin by eukaryotic sequence-specific transcription factors (TFs) is integral to the regulation of gene expression but the mechanism of nuclear exploration has remained obscure. Here we use multicolor single-molecule fluorescence resonance energy transfer and single-particle imaging to track the diffusion of purified Drosophila GAGA factor (GAF) on DNA and nucleosomes. Monomeric GAF DNA-binding domain (DBD) bearing one zinc finger finds its cognate site through one-dimensional (1D) or three-dimensional (3D) diffusion on bare DNA and rapidly slides back and forth between naturally clustered motifs for seconds before dissociation.

A competitive regulatory mechanism of the Chd1 remodeler is integral to distorting nucleosomal DNA.

The Chd1 chromatin remodeler repositions nucleosomes into evenly spaced arrays, a characteristic of most eukaryotic genes. Here we show that the yeast Chd1 remodeler requires two activating segments to distort nucleosomal DNA into an A-form-like conformation, a critical first step in nucleosome sliding. As shown by cryo-electron microscopy, these two activating segments together pack against the ATPase motor, where they are poised to stabilize the central ATPase cleft.

Androgens mediate sexual dimorphism in Pilarowski-Bjornsson Syndrome.

Sex-specific penetrance in autosomal dominant Mendelian conditions is largely understudied. The neurodevelopmental disorder Pilarowski-Bjornsson syndrome (PILBOS) was initially described in females. Here, we describe the clinical and genetic characteristics of the largest PILBOS cohort to date, showing that both sexes can exhibit PILBOS features, although males are overrepresented.

Mechanistic insights into INO80-type chromatin remodelers.

Chromatin remodelers are adenosine triphosphate (ATP)-driven enzymes that physically reorganize nucleosomes, the basic packaging unit of all eukaryotic chromosomes. INO80, SWR1/SRCAP, and TIP60 are large multisubunit remodelers that share similar components yet have distinct biochemical and biological functions. This review summarizes key architectural features of these complexes and how they engage DNA, nucleosomes, and hexasomes to carry out their tasks.

Native nucleosome-positioning elements for the investigation of nucleosome repositioning.

Nucleosome repositioning is essential for establishing nucleosome-depleted regions (NDRs) to initiate transcription. This process has been extensively studied using structural, biochemical, and single-molecule approaches, which require homogenously positioned nucleosomes. This is often achieved using the Widom 601 sequence, a highly efficient nucleosome positioning element (NPE) selected for its unusually strong binding to the H3-H4 histone tetramer.

H3K56 acetylation regulates chromatin maturation following DNA replication.

Following DNA replication, the newly reassembled chromatin is disorganized and must mature to its steady state to maintain both genome and epigenome integrity. However, the regulatory mechanisms governing this critical process remain poorly understood. Here, we show that histone H3K56 acetylation (H3K56ac), a mark on newly-synthesized H3, facilitates the remodeling of disorganized nucleosomes in nascent chromatin, and its removal at the subsequent G2/M phase of the cell cycle marks the completion of chromatin maturation.

Bi-directional nucleosome sliding by the Chd1 chromatin remodeler integrates intrinsic sequence-dependent and ATP-dependent nucleosome positioning.

Chromatin remodelers use a helicase-type ATPase motor to shift DNA around the histone core. Although not directly reading out the DNA sequence, some chromatin remodelers exhibit a sequence-dependent bias in nucleosome positioning, which presumably reflects properties of the DNA duplex. Here, we show how nucleosome positioning by the Chd1 remodeler is influenced by local DNA perturbations throughout the nucleosome footprint.

GAGA zinc finger transcription factor searches chromatin by 1D-3D facilitated diffusion.

To elucidate how eukaryotic sequence-specific transcription factors (TFs) search for gene targets on chromatin, we used multi-color smFRET and single-particle imaging to track the diffusion of purified GAGA-Associated Factor (GAF) on DNA and nucleosomes. Monomeric GAF DNA-binding domain (DBD) bearing one zinc finger finds its cognate site by 1D or 3D diffusion on bare DNA and rapidly slides back-and-forth between naturally clustered motifs for seconds before escape. Multimeric, full-length GAF also finds clustered motifs on DNA by 1D-3D diffusion, but remains locked on target for longer periods.

In Vitro Mapping of Nucleosome Positions at Base-Pair Resolution Using Ortho-Phenanthroline.

The positions of nucleosomes along genomic DNA play a role in defining patterns of gene expression and chromatin organization. Determination of nucleosome positions in vivo and in vitro, as revealed by the locations of histones on DNA, has provided insight into mechanisms of nucleosome sliding, spacing, assembly, and disassembly. Here, we describe methods for the in vitro determination of histone-DNA contacts at base-pair (bp) resolution.

Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state.

Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. Here we report a complex of the Chd1 remodeler bound to a nucleosome in a nucleotide-free state, determined by cryo-EM to 2.3 Å resolution.

Reb1, Cbf1, and Pho4 Bias Histone Sliding and Deposition Away from Their Binding Sites.

In transcriptionally active genes, nucleosome positions in promoters are regulated by nucleosome-displacing factors (NDFs) and chromatin-remodeling enzymes. Depletion of NDFs or the RSC chromatin remodeler shrinks or abolishes the nucleosome-depleted regions (NDRs) in promoters, which can suppress gene activation and result in cryptic transcription. Despite their vital cellular functions, how the action of chromatin remodelers may be directly affected by site-specific binding factors like NDFs is poorly understood.

Autoinhibitory elements of the Chd1 remodeler block initiation of twist defects by destabilizing the ATPase motor on the nucleosome.

Chromatin remodelers are ATP (adenosine triphosphate)-powered motors that reposition nucleosomes throughout eukaryotic chromosomes. Remodelers possess autoinhibitory elements that control the direction of nucleosome sliding, but underlying mechanisms of inhibition have been unclear. Here, we show that autoinhibitory elements of the yeast Chd1 remodeler block nucleosome sliding by preventing initiation of twist defects.

Biophysics of Chromatin Remodeling.

As primary carriers of epigenetic information and gatekeepers of genomic DNA, nucleosomes are essential for proper growth and development of all eukaryotic cells. Although they are intrinsically dynamic, nucleosomes are actively reorganized by ATP-dependent chromatin remodelers. Chromatin remodelers contain helicase-like ATPase motor domains that can translocate along DNA, and a long-standing question in the field is how this activity is used to reposition or slide nucleosomes.

Reconstitution and Purification of Nucleosomes with Recombinant Histones and Purified DNA.

Nucleosomes are substrates for a broad range of factors, including those involved in transcription or chromosome maintenance/reorganization and enzymes that covalently modify histones. Given the heterogeneous nature of nucleosomes in vivo (i.e.

The Chd1 chromatin remodeler forms long-lived complexes with nucleosomes in the presence of ADP·BeF and transition state analogs.

Chromatin remodelers use helicase-like ATPase domains to reorganize histone-DNA contacts within the nucleosome. Like other remodelers, the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler repositions nucleosomes by altering DNA topology at its internal binding site on the nucleosome, coupling different degrees of DNA twist and DNA movement to distinct nucleotide-bound states of the ATPase motor. In this work, we used a competition assay to study how variations in the bound nucleotide, Chd1, and the nucleosome substrate affect stability of Chd1-nucleosome complexes.

Uncovering a New Step in Sliding Nucleosomes.

Chromatin remodelers are ATP-driven motors that pump double-stranded DNA around the histone core of the nucleosome. Recent work by Chen and coworkers (Li et al., Nature, 2019 and Yan et al.

Asymmetry between the two acidic patches dictates the direction of nucleosome sliding by the ISWI chromatin remodeler.

The acidic patch is a functionally important epitope on each face of the nucleosome that affects chromatin remodeling. Although related by 2-fold symmetry of the nucleosome, each acidic patch is uniquely positioned relative to a bound remodeler. An open question is whether remodelers are distinctly responsive to each acidic patch.

Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling.

ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET.

The ATPase motor of the Chd1 chromatin remodeler stimulates DNA unwrapping from the nucleosome.

Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-.

A twist defect mechanism for ATP-dependent translocation of nucleosomal DNA.

As superfamily 2 (SF2)-type translocases, chromatin remodelers are expected to use an inchworm-type mechanism to walk along DNA. Yet how they move DNA around the histone core has not been clear. Here we show that a remodeler ATPase motor can shift large segments of DNA by changing the twist and length of nucleosomal DNA at superhelix location 2 (SHL2).

The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer.

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome.

Missense variants in the chromatin remodeler are associated with neurodevelopmental disability.

Background: The list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler have been found in several large-scale sequencing efforts focused on uncovering the genetic aetiology of autism.

Objectives: To explore whether variants in are associated with a human phenotype.