Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001

[1]  D. Jeste,et al.  Reduced Purkinje cell density in Huntington's disease , 1984, Experimental Neurology.

[2]  J. B. Martin,et al.  Selective sparing of a class of striatal neurons in Huntington's disease. , 1985, Science.

[3]  R. Tjian,et al.  Synergistic activation by the glutamine-rich domains of human transcription factor Sp1 , 1989, Cell.

[4]  J. Penney,et al.  The functional anatomy of basal ganglia disorders , 1989, Trends in Neurosciences.

[5]  Manish S. Shah,et al.  A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes , 1993, Cell.

[6]  R. Albin,et al.  Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues , 1993, Nature Genetics.

[7]  R. Tjian,et al.  A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S. Floresco,et al.  Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes , 1995, Cell.

[9]  Virginia E. Papaioannou,et al.  Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue , 1995, Nature Genetics.

[10]  S. W. Davies,et al.  Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. , 1997, Science.

[11]  Hans Lehrach,et al.  Huntingtin-Encoded Polyglutamine Expansions Form Amyloid-like Protein Aggregates In Vitro and In Vivo , 1997, Cell.

[12]  Steven Finkbeiner,et al.  Huntingtin Acts in the Nucleus to Induce Apoptosis but Death Does Not Correlate with the Formation of Intranuclear Inclusions , 1998, Cell.

[13]  René Hen,et al.  Reversal of Neuropathology and Motor Dysfunction in a Conditional Model of Huntington's Disease , 2000, Cell.

[14]  S. Lindquist,et al.  Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. MacDonald,et al.  Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. , 2000, Human molecular genetics.

[16]  A. Monaco,et al.  A forkhead-domain gene is mutated in a severe speech and language disorder , 2001, Nature.

[17]  D. Rubinsztein,et al.  Wild type huntingtin reduces the cellular toxicity of mutant huntingtin in mammalian cell models of Huntington's disease , 2001, Journal of medical genetics.

[18]  Rahul S. Rajan,et al.  Specificity in intracellular protein aggregation and inclusion body formation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C A Ross,et al.  Interference by Huntingtin and Atrophin-1 with CBP-Mediated Transcription Leading to Cellular Toxicity , 2001, Science.

[20]  Richard I. Morimoto,et al.  Polyglutamine protein aggregates are dynamic , 2002, Nature Cell Biology.

[21]  A. Monaco,et al.  Molecular evolution of FOXP2, a gene involved in speech and language , 2002, Nature.

[22]  Dimitri Krainc,et al.  Sp1 and TAFII130 Transcriptional Activity Disrupted in Early Huntington's Disease , 2002, Science.

[23]  D. Rubinsztein.  Lessons from animal models of Huntington's disease. , 2002, Trends in genetics : TIG.

[24]  Judith P. Johnson,et al.  Identification of the human PHLDA1/TDAG51 gene: down-regulation in metastatic melanoma contributes to apoptosis resistance and growth deregulation. , 2002, Cancer research.

[25]  Kaoru Takahashi,et al.  Expression of Foxp2, a gene involved in speech and language, in the developing and adult striatum , 2003, Journal of neuroscience research.

[26]  G. Bates,et al.  Huntingtin aggregation and toxicity in Huntington's disease , 2003, The Lancet.

[27]  H. Katoh,et al.  A Role of Rnd1 GTPase in Dendritic Spine Formation in Hippocampal Neurons , 2003, The Journal of Neuroscience.

[28]  E. Simpson,et al.  Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. , 2003, Human molecular genetics.

[29]  Wu Mx.  Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis. , 2003 .

[30]  Christine Van Broeckhoven,et al.  Pathogenesis of polyglutamine disorders: aggregation revisited. , 2003, Human molecular genetics.

[31]  Fumiaki Tanaka,et al.  Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. , 2003, Human molecular genetics.

[32]  E. Seeberg,et al.  Mutant Huntingtin Impairs Axonal Trafficking in Mammalian Neurons In Vivo and In Vitro , 2004, Molecular and Cellular Biology.

[33]  M. Wu.  Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis , 2004, Apoptosis.

[34]  Mark R. Segal,et al.  Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death , 2004, Nature.

[35]  Fabrice P Cordelières,et al.  Huntingtin Controls Neurotrophic Support and Survival of Neurons by Enhancing BDNF Vesicular Transport along Microtubules , 2004, Cell.

[36]  J. Littleton,et al.  Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[37]  R. Tjian,et al.  In Vitro Analysis of Huntingtin-Mediated Transcriptional Repression Reveals Multiple Transcription Factor Targets , 2005, Cell.

[38]  Elena Cattaneo,et al.  Normal huntingtin function: an alternative approach to Huntington's disease , 2005, Nature Reviews Neuroscience.

[39]  M. Hayden,et al.  Absence of behavioral abnormalities and neurodegeneration in vivo despite widespread neuronal huntingtin inclusions. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[40]  J. Buxbaum,et al.  Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Zeitlinger,et al.  Polycomb complexes repress developmental regulators in murine embryonic stem cells , 2006, Nature.

[42]  J. Elf,et al.  Probing Transcription Factor Dynamics at the Single-Molecule Level in a Living Cell , 2007, Science.

[43]  R. Myers,et al.  Wild-type huntingtin participates in protein trafficking between the Golgi and the extracellular space. , 2007, Human molecular genetics.

[44]  J. Lippincott-Schwartz,et al.  High-density mapping of single-molecule trajectories with photoactivated localization microscopy , 2008, Nature Methods.

[45]  A. Sergé,et al.  Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes , 2008, Nature Methods.

[46]  Clifford A. Meyer,et al.  Model-based Analysis of ChIP-Seq (MACS) , 2008, Genome Biology.

[47]  Steve D. M. Brown,et al.  Impaired Synaptic Plasticity and Motor Learning in Mice with a Point Mutation Implicated in Human Speech Deficits , 2008, Current Biology.

[48]  H. Paulson,et al.  Polyglutamine neurodegeneration: protein misfolding revisited , 2008, Trends in Neurosciences.

[49]  T. Misteli,et al.  Transcription dynamics. , 2009, Molecular cell.

[50]  Johannes Schwarz,et al.  A Humanized Version of Foxp2 Affects Cortico-Basal Ganglia Circuits in Mice , 2009, Cell.

[51]  Lior Pachter,et al.  Sequence Analysis , 2020, Definitions.

[52]  Cory Y. McLean,et al.  GREAT improves functional interpretation of cis-regulatory regions , 2010, Nature Biotechnology.

[53]  B. Pugh,et al.  Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution , 2011, Cell.

[54]  G. Bates,et al.  Altered chromatin architecture underlies progressive impairment of the heat shock response in mouse models of Huntington disease. , 2011, The Journal of clinical investigation.

[55]  C. Ross,et al.  Huntington's disease: from molecular pathogenesis to clinical treatment , 2011, The Lancet Neurology.

[56]  Jimin Pei,et al.  Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels , 2012, Cell.

[57]  Daniel Sage,et al.  Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation , 2012, BMC Bioinformatics.

[58]  Significantly differential diffusion of neuropathological aggregates in the brain of transgenic mice carrying N-terminal mutant huntingtin fused with green fluorescent protein , 2012, Brain Structure and Function.

[59]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[60]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[61]  J. McNally,et al.  A benchmark for chromatin binding measurements in live cells , 2012, Nucleic acids research.

[62]  Alexander Y Katsov,et al.  fast multicolor 3 d imaging using aberration-corrected multifocus microscopy , 2012 .

[63]  M. Davidson,et al.  Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination , 2012, Proceedings of the National Academy of Sciences.

[64]  Lucien E. Weiss,et al.  Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species , 2012, Scientific Reports.

[65]  J. Taylor,et al.  Huntingtin Fragments and SOD1 Mutants Form Soluble Oligomers in the Cell , 2012, PloS one.

[66]  Yongdeng Zhang,et al.  Rational design of true monomeric and bright photoactivatable fluorescent proteins , 2012, Nature Methods.

[67]  S. Shaham,et al.  PolyQ disease: misfiring of a developmental cell death program? , 2013, Trends in cell biology.

[68]  J. McNally,et al.  Quantifying transcription factor kinetics: At work or at play? , 2013, Critical reviews in biochemistry and molecular biology.

[69]  X. Xie,et al.  Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells , 2013, Nature Methods.

[70]  Ignacio Izeddin,et al.  Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells , 2013, Science.

[71]  Udo Rüb,et al.  Degeneration of the Cerebellum in Huntington's Disease (HD): Possible Relevance for the Clinical Picture and Potential Gateway to Pathological Mechanisms of the Disease Process , 2013, Brain pathology.

[72]  R. Morimoto,et al.  Huntington's disease: underlying molecular mechanisms and emerging concepts. , 2013, Trends in biochemical sciences.

[73]  S. Snyder,et al.  Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington’s disease , 2014, Nature.

[74]  M. Dahan,et al.  Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus , 2014, eLife.

[75]  Wesley R. Legant,et al.  Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells , 2014, Cell.

[76]  R. Ratan,et al.  Transcriptional dysregulation in Huntington's disease: a failure of adaptive transcriptional homeostasis. , 2014, Drug discovery today.

[77]  J. Frydman,et al.  Super-Resolution Fluorescence of Huntingtin Reveals Growth of Globular Species into Short Fibers and Coexistence of Distinct Aggregates , 2014, ACS chemical biology.

[78]  J. Tinevez,et al.  TNF and IL-1 exhibit distinct ubiquitin requirements for inducing NEMO–IKK supramolecular structures , 2014, The Journal of cell biology.

[79]  Wesley R. Legant,et al.  3D imaging of Sox2 enhancer clusters in embryonic stem cells , 2014, eLife.

[80]  J. Workman,et al.  The expanding role for chromatin and transcription in polyglutamine disease. , 2014, Current opinion in genetics & development.

[81]  Zia Choudhry,et al.  The mechanism of degeneration of striatal neuronal subtypes in Huntington disease , 2014, Annals of neurosciences.

[82]  S. Tabrizi,et al.  Cerebellar abnormalities in Huntington's disease: A role in motor and psychiatric impairment? , 2014, Movement disorders : official journal of the Movement Disorder Society.

[83]  Wesley R. Legant,et al.  Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution , 2014, Science.

[84]  E. Betzig,et al.  Imaging live-cell dynamics and structure at the single-molecule level. , 2015, Molecular cell.

[85]  Lucien E. Weiss,et al.  Delayed emergence of subdiffractionsized mutant huntingtin fibrils following inclusion body formation , 2015, Quarterly Reviews of Biophysics.

[86]  Claire H. Michel,et al.  ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function , 2015, Neuron.

[87]  Maxime Dahan,et al.  InferenceMAP: mapping of single-molecule dynamics with Bayesian inference , 2015, Nature Methods.

[88]  Marco Y. Hein,et al.  A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation , 2015, Cell.

[89]  Ji-feng Guo,et al.  Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice. , 2015, Cell reports.

[90]  J. J. Macklin,et al.  A general method to improve fluorophores for live-cell and single-molecule microscopy , 2014, Nature Methods.

[91]  Yonghao Yu,et al.  The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei , 2015, Cell.

[92]  Timothy D. Craggs,et al.  Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles , 2015, Molecular cell.

[93]  A. Kanagaraj,et al.  Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization , 2015, Cell.

[94]  Brian P. Mehl,et al.  Bright photoactivatable fluorophores for single-molecule imaging , 2016, Nature Methods.

[95]  M. Mann,et al.  Cytoplasmic protein aggregates interfere with nucleocytoplasmic transport of protein and RNA , 2016, Science.