Energy-driven genome regulation by ATP-dependent chromatin remodellers.

The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism. Whether remodeller activities mainly catalyse nucleosome dynamics or accurately co-determine nucleosome organization remained unclear. In this Review, we discuss the emerging mechanisms of chromatin remodelling: dynamic remodeller architectures and their interactions, the inner workings of the ATPase cycle, allosteric regulation and pathological dysregulation. Recent mechanistic insights argue for a decisive role of remodellers in the energy-driven self-organization of chromatin, which enables both stability and plasticity of genome regulation - for example, during development and stress. Different remodellers, such as members of the SWI/SNF, ISWI, CHD and INO80 families, process (epi)genetic information through specific mechanisms into distinct functional outputs. Combinatorial assembly of remodellers and their interplay with histone modifications, histone variants, DNA sequence or DNA-bound transcription factors regulate nucleosome mobilization or eviction or histone exchange. Such input-output relationships determine specific nucleosome positions and compositions with distinct DNA accessibilities and mediate differential genome regulation. Finally, remodeller genes are often mutated in diseases characterized by genome dysregulation, notably in cancer, and we discuss their physiological relevance.

Hexasome-INO80 complex reveals structural basis of noncanonical nucleosome remodeling.

Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.

Structural basis for TBP displacement from TATA box DNA by the Swi2/Snf2 ATPase Mot1.

The Swi2/Snf2 family transcription regulator Modifier of Transcription 1 (Mot1) uses adenosine triphosphate (ATP) to dissociate and reallocate the TATA box-binding protein (TBP) from and between promoters. To reveal how Mot1 removes TBP from TATA box DNA, we determined cryogenic electron microscopy structures that capture different states of the remodeling reaction. The resulting molecular video reveals how Mot1 dissociates TBP in a process that, intriguingly, does not require DNA groove tracking. Instead, the motor grips DNA in the presence of ATP and swings back after ATP hydrolysis, moving TBP to a thermodynamically less stable position on DNA. Dislodged TBP is trapped by a chaperone element that blocks TBP's DNA binding site. Our results show how Swi2/Snf2 proteins can remodel protein-DNA complexes through DNA bending without processive DNA tracking and reveal mechanistic similarities to RNA gripping DEAD box helicases and RIG-I-like immune sensors.

A hold-and-feed mechanism drives directional DNA loop extrusion by condensin.

Structural maintenance of chromosomes (SMC) protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop transiently in two separate chambers. Single-molecule imaging and cryo-electron microscopy suggest a putative power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon adenosine triphosphate binding, whereas the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of "motor" and "anchor" chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the SMC reaction cycle determines the directionality of DNA loop extrusion.

Cathepsin S Alterations Induce a Tumor-Promoting Immune Microenvironment in Follicular Lymphoma.

Tumor cells orchestrate their microenvironment. Here, we provide biochemical, structural, functional, and clinical evidence that Cathepsin S (CTSS) alterations induce a tumor-promoting immune microenvironment in follicular lymphoma (FL). We found CTSS mutations at Y132 in 6% of FL (19/305). Another 13% (37/286) had CTSS amplification, which was associated with higher CTSS expression. CTSS Y132 mutations lead to accelerated autocatalytic conversion from an enzymatically inactive profrom to active CTSS and increased substrate cleavage, including CD74, which regulates major histocompatibility complex class II (MHC class II)-restricted antigen presentation. Lymphoma cells with hyperactive CTSS more efficiently activated antigen-specific CD4+ T cells in vitro. Tumors with hyperactive CTSS showed increased CD4+ T cell infiltration and proinflammatory cytokine perturbation in a mouse model and in human FLs. In mice, this CTSS-induced immune microenvironment promoted tumor growth. Clinically, patients with CTSS-hyperactive FL had better treatment outcomes with standard immunochemotherapies, indicating that these immunosuppressive regimens target both the lymphoma cells and the tumor-promoting immune microenvironment.

Near-Complete Structure and Model of Tel1ATM from Chaetomium thermophilum Reveals a Robust Autoinhibited ATP State.

Tel1 (ATM in humans) is a large kinase that resides in the cell in an autoinhibited dimeric state and upon activation orchestrates the cellular response to DNA damage. We report the structure of an endogenous Tel1 dimer from Chaetomium thermophilum. Major parts are at 2.8 Å resolution, including the kinase active site with ATPγS bound, and two different N-terminal solenoid conformations are at 3.4 Å and 3.6 Å, providing a side-chain model for 90% of the Tel1 polypeptide. We show that the N-terminal solenoid has DNA binding activity, but that its movements are not coupled to kinase activation. Although ATPγS and catalytic residues are poised for catalysis, the kinase resides in an autoinhibited state. The PIKK regulatory domain acts as a pseudo-substrate, blocking direct access to the site of catalysis. The structure allows mapping of human cancer mutations and defines mechanisms of autoinhibition at near-atomic resolution.

Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers1-3 such as the 15-subunit INO80 complex 4 . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA5-8. The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA+ ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data 8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.

Single-molecule nucleosome remodeling by INO80 and effects of histone tails.

Genome maintenance and integrity requires continuous alterations of the compaction state of the chromatin structure. Chromatin remodelers, among others the INO80 complex, help organize chromatin by repositioning, reshaping, or evicting nucleosomes. We report on INO80 nucleosome remodeling, assayed by single-molecule Foerster resonance energy transfer on canonical nucleosomes as well as nucleosomes assembled from tailless histones. Nucleosome repositioning by INO80 is a processively catalyzed reaction. During the initiation of remodeling, probed by the INO80 bound state, the nucleosome reveals structurally heterogeneous states for tailless nucleosomes (in contrast to wild-type nucleosomes). We, therefore, propose an altered energy landscape for the INO80-mediated nucleosome sliding reaction in the absence of histone tails.

A Poly-ADP-Ribose Trigger Releases the Auto-Inhibition of a Chromatin Remodeling Oncogene.

DNA damage triggers chromatin remodeling by mechanisms that are poorly understood. The oncogene and chromatin remodeler ALC1/CHD1L massively decompacts chromatin in vivo yet is inactive prior to DNA-damage-mediated PARP1 induction. We show that the interaction of the ALC1 macrodomain with the ATPase module mediates auto-inhibition. PARP1 activation suppresses this inhibitory interaction. Crucially, release from auto-inhibition requires a poly-ADP-ribose (PAR) binding macrodomain. We identify tri-ADP-ribose as a potent PAR-mimic and synthetic allosteric effector that abrogates ATPase-macrodomain interactions, promotes an ungated conformation, and activates the remodeler's ATPase. ALC1 fragments lacking the regulatory macrodomain relax chromatin in vivo without requiring PARP1 activation. Further, the ATPase restricts the macrodomain's interaction with PARP1 under non-DNA damage conditions. Somatic cancer mutants disrupt ALC1's auto-inhibition and activate chromatin remodeling. Our data show that the NAD+-metabolite and nucleic acid PAR triggers ALC1 to drive chromatin relaxation. Modular allostery in this oncogene tightly controls its robust, DNA-damage-dependent activation.

Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1.

Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.

Solution structures of the two PBZ domains from human APLF and their interaction with poly(ADP-ribose).

Addition of poly(ADP-ribose) (PAR) is an important post-translational modification in higher eukaryotes. Several DNA repair and checkpoint proteins possess specific PAR-binding zinc-finger (PBZ) modules critical for function. Here, we present solution structures of the two PBZ modules of aprataxin and PNK-like factor (APLF), revealing a novel type of zinc finger. By combining in vivo PAR-binding data with NMR interaction data using PAR fragments, we propose a structural basis for PBZ-PAR recognition.