Structural and functional characterization of the INO80 chromatin remodelling complex

DNA is the carrier of the genetic information in all kingdoms of life. Cells face the challenge to pack DNA and ensure its integrity on the one hand while enabling access to the genetic code on the other hand. This holds in particular true for eukaryotes, whose genomes are typically larger than those of prokaryotes and organized in multiple linear DNA molecules, termed chromosomes, within the nuclear envelope. Their genetic information is stored as a nucleoprotein complex referred to as chromatin, in which DNA is associated with histone proteins. It compacts DNA and at the same time provides an elaborate platform to regulate access to the genetic code. Various fundamental cellular processes depend on this access and thus are regulated by the organization of chromatin, such as transcription, cell division, cell differentiation and DNA repair. The fundamental unit of chromatin is the nucleosome core particle, in which 147 bp of DNA are wrapped around an octamer of the four core histone proteins (H2A, H2B, H3 and H4) or variants thereof, giving rise to a disk-shaped particle. The nucleosome core particle originated from archaea. These possess one or two histone proteins, which are orthologous to eukaryotic histones and assemble with DNA in an overall similar fashion. Being the fundamental unit of chromatin, the formation, disassembly, localization and composition of the individual nucleosome core particles directly impacts the chromatin landscape and therefore gene regulation. These actions are carried out by chromatin remodelling complexes (‘remodellers’). The catalytic core of all remodellers is a Snf2-type ATPase, which converts the energy of ATP hydrolysis in DNA translocation. Based on flanking domains and additional subunits, remodellers can be grouped into four families: ISWI, CHD, SWI/SNF and INO80. While ISWI and CHD carry out their function as small complexes or even as single subunits, remodellers of the SWI/SNF and INO80 families form multi-subunit complexes in the megadalton range. In the past twenty years, several hallmark studies characterized the biological functions of these multi-subunit complexes and analyzed their composition and architecture. However, insights on a detailed structural level into how the individual subunits cooperate remained elusive, mainly due to technical limitations. These could partly be overcome in the past years, especially by the advent of high-resolution cryogenic electron microscopy. This thesis analyzes the INO80 chromatin remodelling complex (INO80), the founding member of the INO80 family, from a structural and functional perspective with an emphasis on its action on the nucleosome core particle. INO80 translocates DNA around the nucleosome core particle and spaces nucleosomes to form genic arrays. The presented results reveal, how the evolutionarily conserved subunits of INO80 interact with the nucleosome and catalyze DNA translocation in a coordinated fashion. A cryo-EM structure of the core module of INO80 bound to the nucleosome core particle demonstrates that the ATPase domain and the actin fold of Arp5 bind nucleosomal DNA at SHL -6 and SHL -3, respectively, while the insert domain of Arp5 contacts the acidic patch. The Rvb1/2 heterohexamer connects these subunits without forming major nucleosome contacts. This arrangement provides valuable information about the mechano-chemical catalysis cycle of INO80, in which the ATPase domain acts as a motor, Arp5 as a counter grip and the Rvb1/2 ring as a stator element. The ATPase pumps DNA inside the nucleosome core particle against Arp5, which leads to a DNA strain. Once sufficient force is generated, the counter grip is released and DNA translocation occurs. Thus, these results explain the biochemically and biophysically observed step size of 10 – 20 bp of DNA translocation catalyzed by INO80. The X-ray structure of the Arp8 module in combination with biochemical data shows that the module binds outside the nucleosome core particle to extranucleosomal DNA. Arp8, actin and Arp4 organize the HSA domain of Ino80 in a way that a number of conserved and positively charged lysine and arginine residues interact with entry DNA ahead of the ATPase domain. This interaction is crucial for the catalysis of DNA translocation by INO80. The combination of these structures leads to a composite model of the evolutionarily conserved subunits of INO80, which is supported by a more recent cryo-EM structure. It suggests that the Arp8 module prevents DNA residing in a transition state between the ATPase and Arp5 from slipping back. Moreover, the Arp8 module could also act as a molecular ruler as its footprint matches the distance between two nucleosome core particles in genic arrays formed by INO80. Small molecule analysis reveals that histone tails regulate nucleosome invasion by INO80. They constitute a regulatory barrier and constrain conformations of nucleosome-bound INO80

Genome information processing by the INO80 chromatin remodeler positions nucleosomes [preprint]

The fundamental molecular determinants by which ATP-dependent chromatin remodelers organize nucleosomes across eukaryotic genomes remain largely elusive. Here, chromatin reconstitutions on physiological, whole-genome templates reveal how remodelers read and translate genomic information into nucleosome positions. Using the yeast genome and the multi-subunit INO80 remodeler as a paradigm, we identify DNA shape/mechanics encoded signature motifs as sufficient for nucleosome positioning and distinct from known DNA sequence preferences of histones. INO80 processes such information through an allosteric interplay between its core- and Arp8-modules that probes mechanical properties of nucleosomal and linker DNA. At promoters, INO80 integrates this readout of DNA shape/mechanics with a readout of co-evolved sequence motifs via interaction with general regulatory factors bound to these motifs. Our findings establish a molecular mechanism for robust and yet adjustable +1 nucleosome positioning and, more generally, remodelers as information processing hubs that enable active organization and allosteric regulation of the first level of chromatin

Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes

Crosstalk between Medulloblastoma Cells and Endothelium Triggers a Strong Chemotactic Signal Recruiting T Lymphocytes to the Tumor Microenvironment

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)” is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant “Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES).” This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications

Connectivity of the Primate Superior Colliculus Mapped by Concurrent Microstimulation and Event-Related fMRI

Background: Neuroanatomical studies investigating the connectivity of brain areas have heretofore employed procedures in which chemical or viral tracers are injected into an area of interest, and connected areas are subsequently identified using histological techniques. Such experiments require the sacrifice of the animals and do not allow for subsequent electrophysiological studies in the same subjects, rendering a direct investigation of the functional properties of anatomically identified areas impossible. Methodology/Principal Findings: Here, we used a combination of microstimulation and fMRI in an anesthetized monkey preparation to study the connectivity of the superior colliculus (SC). Microstimulation of the SC resulted in changes in the blood oxygenation level-dependent (BOLD) signals in the SC and in several cortical and subcortical areas consistent with the known connectivity of the SC in primates. Conclusions/Significance: These findings demonstrates that the concurrent use of microstimulation and fMRI can be used to identify brain networks for further electrophysiological or fMRI investigation

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Dissecting the Shared Genetic Architecture of Suicide Attempt, Psychiatric Disorders, and Known Risk Factors

Background Suicide is a leading cause of death worldwide, and nonfatal suicide attempts, which occur far more frequently, are a major source of disability and social and economic burden. Both have substantial genetic etiology, which is partially shared and partially distinct from that of related psychiatric disorders. Methods We conducted a genome-wide association study (GWAS) of 29,782 suicide attempt (SA) cases and 519,961 controls in the International Suicide Genetics Consortium (ISGC). The GWAS of SA was conditioned on psychiatric disorders using GWAS summary statistics via multitrait-based conditional and joint analysis, to remove genetic effects on SA mediated by psychiatric disorders. We investigated the shared and divergent genetic architectures of SA, psychiatric disorders, and other known risk factors. Results Two loci reached genome-wide significance for SA: the major histocompatibility complex and an intergenic locus on chromosome 7, the latter of which remained associated with SA after conditioning on psychiatric disorders and replicated in an independent cohort from the Million Veteran Program. This locus has been implicated in risk-taking behavior, smoking, and insomnia. SA showed strong genetic correlation with psychiatric disorders, particularly major depression, and also with smoking, pain, risk-taking behavior, sleep disturbances, lower educational attainment, reproductive traits, lower socioeconomic status, and poorer general health. After conditioning on psychiatric disorders, the genetic correlations between SA and psychiatric disorders decreased, whereas those with nonpsychiatric traits remained largely unchanged. Conclusions Our results identify a risk locus that contributes more strongly to SA than other phenotypes and suggest a shared underlying biology between SA and known risk factors that is not mediated by psychiatric disorders.Peer reviewe

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder