Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the polyglutamine (polyQ) expansion in huntingtin (HTT) protein. The challenge of obtaining full-length HTT proteins with high purity limits the understanding of the HTT protein function. Here, we provide a protocol to generate and purify full-length recombinant human HTT proteins with various polyQ lengths, which is key to investigate the biochemical function of HTT proteins and the molecular mechanism underlying HD pathology. For complete details on the use and execution of this protocol, please refer to Jung et al. (2020).

In this review, we summarize the available published information on the neuroprotective effects of increasing nicotinamide adenine dinucleotide (NAD + ) levels in Huntington’s disease models. We discuss the rationale of potential therapeutic benefit of administering nicotinamide riboside (NR), a safe and effective NAD + precursor. We discuss the agonistic effect on the Sirtuin1-PGC-1α-PPAR pathway as well as Sirtuin 3, which converge in improving mitochondrial function, decreasing ROS production and ameliorating bioenergetics deficits. Also, we discuss the potential synergistic effect of increasing NAD+ combined with PARPs inhibitors, as a clinical therapeutic option not only in HD, but other neurodegenerative conditions.

The polyglutamine expansion in huntingtin protein causes Huntington’s disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The polyglutamine expansion in huntingtin protein causes Huntington’s disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single-particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical α-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases α-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease. https://doi.org/10.7554/eLife.11184.001 eLife digest Huntington’s disease is an inherited disorder that occurs in adulthood and sometimes in children. It causes progressive damage to the brain and people with the condition develop memory loss, movement difficulties, confusion, and other symptoms of mental decline. Eventually, the disease leads to death. Mutations in the gene that encodes a protein called huntingtin cause Huntington’s disease. Individuals who inherit just one copy of the mutated gene develop the condition. No treatments currently exist that can slow or stop disease progression. Genetic and molecular studies are beginning to shed light on how mutations in the gene encoding huntingtin cause the disease. Normally, the protein has a section near its tail end made up of the amino acid glutamine repeated around 23 times. Mutations that increase the number of glutamines to more than 38 cause Huntington’s disease. The more extra glutamines there are in this region of the protein, the earlier in life the disease symptoms begin. But it was not clear how these extra glutamines near the tail of huntingtin affect the structure and behavior of a protein that is more than 3,000 amino acids long. Now, Vijayvargia et al. have revealed why the tail end of huntingtin is so important. Several biophysical methods were used to determine the three-dimensional structure of the huntingtin protein. These methods revealed that the protein folds up into a hollow sphere and that its tail end is able to interact with the entire length of the protein and physically touches its opposite end. To see this in more detail, Vijayvargia et al. used another experimental technique called crosslinking mass spectrometry to confirm which parts of the huntingtin protein are in close contact with each other. Together with the structural data, these experiments suggest that the stretch of glutamines is in the position to bring about subtle, but widespread, changes throughout the huntingtin protein. That is to say, that having more glutamines slightly changes the curve of the sphere and alters the way different parts of the protein interact. Together the new findings explain why mutations that alter the tail of huntingtin affect the rest of the protein. Further work will now aim to provide a more-detailed structure of the huntingtin protein and to investigate what other roles of huntingtin are affected by the increased number of glutamines in the protein’s tail. These insights may help scientists understand how the mutated protein causes brain decline. https://doi.org/10.7554/eLife.11184.002 Introduction Huntingtin is the entire large protein product (>350 kDa MW) of the Huntingtin gene (HTT previously HD). Huntingtin has a segment of polyglutamine near its amino terminus (Amino-terminal) that is encoded by a polymorphic CAG trinucleotide repeat. If expanded above 38-residues, this mutation causes Huntington’s disease (HD), a dominant neurodegenerative disorder (Huntington's Disease Collaborative Research Group, 1993). The strong correlation between the size of the expanded repeat and the age at diagnosis of HD motor, cognitive and psychiatric symptoms shows that CAG repeat-size is the primary determinant of the rate of the disease progression (Brinkman et al., 1997; Snell et al., 1993). This biological relationship also provides a human patient-based rationale for delineating the HD disease-trigger in studies with an allelic series designed to determine the effects of progressively increasing the size of the mutation. Consistent with genetic studies in distinct CAG-expansion neurodegenerative disorders and CAG knock-in mice that replicate the HD mutation, the mechanism that triggers the disease process that leads to the characteristic vulnerability of striatal neurons in HD is thought to involve a novel gain of function that is conferred on mutant huntingtin by the expanded polyglutamine segment (Gusella and MacDonald, 2000; Nucifora et al., 2001; Trettel et al., 2000). By analogy with other members of the HEAT/HEAT-like (Huntingtin, Elongation factor 3, protein phosphatase 2A, Target of rapamycin 1) repeat family (Andrade and Bork, 1995; Perry and Kleckner, 2003), huntingtin is likely a HEAT domain solenoid that functions as a mechanical scaffold for multi-member complexes (Grinthal et al., 2010; Takano and Gusella, 2002). Huntingtin’s large size and predicted predominant HEAT/HEAT-like repeat domain structure is well conserved through 500 million years of evolution (Seong et al., 2010). The polyglutamine region is not conserved in some huntingtin orthologues (Seong et al., 2010), implying a role as an extra feature that fine-tunes huntingtin structure and function. Indeed, testing this idea, we have previously demonstrated, with purified recombinant human huntingtins in a cell-free assay, that lengthening the polyglutamine tract quantitatively enhances the basal function of huntingtin in stimulating Polycomb repressive complex 2 (PRC2) histone methyltransferase (Seong et al., 2010). The structures of smaller HEAT/HEAT-like repeat solenoid scaffold proteins, such as PR65/A and Importin β, have been solved to high-resolution, and each has been shown to assume a distinctive extended curvilinear shape determined by the specific stacking characteristics of its HEAT/HEAT-like repeats (Cingolani et al., 1999; Groves et al., 1999). The topology of the huntingtin solenoid is expected to reflect the specific stacking characteristics of α-helical HEAT/HEAT-like repeats that span the molecule. The shape imparted by intramolecular stacking cannot be predicted because of the idiosyncratic nature of HEAT/HEAT-like repeats, which are loosely conserved ~34 amino acid bipartite α-helical units (Takano and Gusella, 2002). Nevertheless this shape must enable modulation by the amino-terminal polyglutamine segment. It seems reasonable that this may involve some structural feature that is critical to huntingtin function. One possibility is structure-dependent post-translational modification. Human huntingtin is phosphorylated, at more than seventy modified serine, threonine and tyrosine residues (Hornbeck et al., 2012). Indeed, a comparison of lines of transgenic modified HTT BAC mice has implicated unique amino-terminal serine phosphorylation in protection against deleterious effects of mutant huntingtin (Gu et al., 2009) and the polyglutamine expansion at the amino-terminal causes a trend of hypo-phosphorylation in all sites, including sites near the carboxyl-terminus (Anne et al., 2007; Schilling et al., 2006; Warby et al., 2005), indirectly implying a long-range impact of the amino-terminal region on huntingtin structure and function. In order to solve the apparent puzzle of how huntingtin’s solenoid structure may enable quantitative or qualitative (or both) modulation of huntingtin function, according to the size of the amino-terminal polyglutamine tract, we have extended initial observations showing a likely flexible α-helical structure by conducting systematic biophysical and biochemical analyses of members of a panel of highly purified human recombinant huntingtins, with varied lengths of polyglutamine tracts (Fodale et al., 2014; Huang et al., 2015; Li et al., 2006). Results Huntingtin α-helical structure is quantitatively altered with polyglutamine tract size It has been reported previously that purified huntingtin exhibits a predominantly α-helical secondary structure but among studies the impact of polyglutamine size has been inconsistent (Fodale et al., 2014; Huang et al., 2015; Li et al., 2006). To carry out a standardized evaluation, we performed circular dichroism (CD) analysis of a series of recombinant human huntingtins with different polyglutamine tract lengths (Q2-, Q23-, Q46-, Q67-, Q78-huntingtin, respectively) purified to homogeneity (Figure 1—figure supplement 1). The CD spectra (Figure 1B) of all of the huntingtins are consistent with a predominant α-helical secondary structure (Liu and Rost, 2003; Rost et al., 1994) (Figure 1A), with typical minima at 222 and 208 nm and a positive peak at 195 nm, and all exhibited the same irreversible thermal denaturation pattern, with secondary structure stable up to ~38–40°C, a gradual slow denaturation as the temperature is increased to 65–70°C, followed by aggregation and some precipitation (Figure 1C). These results imply the same basic core structure and stability regardless of the size of the expanded polyglutamine segment. Plotting an average of the Mean Residue Ellipticity (MRE) at 222 nm (characteristic of an α-helix) reveals an incremental quantitative effect of lengthening the polyglutamine tract at the amino terminus on the α-helicity of the entire molecule (Figure 1D). Figure 1 with 1 supplement see all Download asset Open asset Huntingtin secondary structure is modulated by the length of polyglutamine tract. (A) Human huntingtin amino acid sequence (Homo sapiens; NP_002102) was analyzed for predicted secondary structure using: NORSp (Liu and Rost, 2003) and PROF (Profile network prediction Heidelberg) (Rost et al.,1994). Stick model of human huntingtin protein (3144 amino acids) was generated depicting the predicted alpha helical (red), random coil (yellow) and beta sheet (grey) regions. The polyglutamine tract in the amino-terminus is indicated in purple. (B) The far UV-wavelength scan at 25°C of these purified huntingtin proteins generates a curve typical of α-helical proteins. (C) Thermal behavior of Q23-, Q46- and Q78-huntingtin. The heat denaturation curves, from 25 to 95°C, of all proteins showed the similar pattern of irreversible thermal denaturation starting their denaturation above 40°C by MRE values at 222 nm. Due to inherent variation caused by inefficient mixing in the cuvette with taking readings every five degrees of heating, their heat denaturation curves were acquired in duplicates. Solid line represents heating to 95°C; dotted line represents cooling from 95°C. (D) An average (MRE) in units of deg.cm2/dmol, at 222 nm wavelength characteristic of an α-helix), from two independent experiments, was plotted against the length of the polyglutamine tract of huntingtin proteins (bars represent mean ± SEM). Temperature was 25°C. https://doi.org/10.7554/eLife.11184.003 3D EM analysis reveals a spherical shape with a central cavity and overlying Amino-terminus We then investigated the proposal that huntingtin’s shape may enable a structural impact of the amino-terminal polyglutamine tract, by performing single-particle electron microscopy (EM) of recombinant human huntingtins with polyglutamine tract lengths of 23- and 78-residues. These were purified to high homogeneity using a gradient purification method with mild crosslinking (GraFix) (Kastner et al., 2008), collecting only the monomer fraction for analysis to eliminate potential contributions from oligomeric structures that would confound interpretation of the results (Figure 2—figure supplement 1). Negative-stained micrographs of these proteins confirmed that the samples were highly homogeneous (Figure 2—figure supplement 2). A total of 10,169 particles were chosen for generating 2D class averages and 30 class averages were used for reconstructing a 3D EM map of Q23-huntingtin (Figure 2—figure supplement 3A). The EM map of Q23-huntingtin at about 30 Å resolution, estimated from Fourier shell correlation analysis, shows that the molecule adopts an overall spherical shape with 130 Å height and 100 Å width (Figure 2A and Figure 2—figure supplement 4). The overall shape of huntingtin was not apparently affected either through crosslinking or by the tag as 2D class averages of huntingtin with no cross linker or without tag also showed a similar spherical shape (Figure 2—figure supplement 3). The outer volume of the structure can be roughly estimated as 861,829 Å3 and a Mathew’s coefficient (VM) of 2.48, assuming that Q23-huntingtin is a sphere with 115 Å diameter with 348 kDa molecular weight. Considering the VM=1.23 for the protein itself, Q23-huntingtin appears to contain a large solvent cavity (up to 50% by volume). Consistent with this estimation, the 3D EM reconstruction of Q23-huntingtin shows a large cavity in the core (Figure 2B). The analysis of negatively-stained Q78-huntingtin (Figure 2C and Figure 2—figure supplement 3B) disclosed a 3D map showing a similar overall spherical shape, with a large cavity in the core (Figure 2D). Although 3D maps were reconstructed de novo without other experimental methods such as random conical tilt, the high similarity of the shapes between Q23- and Q78-huntingtin 3D maps attests that huntingtin has the spherical structure with a cavity (Figure 2—figure supplement 5). Notably, manual superimposition of the 3D maps of Q23-huntingtin and Q78-huntingtin reveals potential differences in the two structures (Figure 2—figure supplement 5), which may reflect technical variation (image processing, stain distribution, sample heterogeneity), in addition to the structural effects of the lengthened polyglutamine segment that were foreshadowed by the altered CD spectra (Figure 1D). Figure 2 with 5 supplements see all Download asset Open asset Three-dimensional reconstruction of negatively-stained Q23- and Q78-huntingtin. (A) 3D EM map of Q23-huntingtin was reconstructed from negatively-stained particles of Q23 monomer separated by GraFix. The resolution was estimated as 33.5 Å at 0.5 FSC. 3D map of Q23-huntingtin is shown in different orientation rotated about the y axis (0°, 90°, 180°, 270°). (B) Sectioned view of 3D EM map of Q23-huntingtin in the same orientation as in A revealing a large cavity inside of Q23-huntingtin. (C) 3D EM map of Q78-huntingin (32.0 Å at 0.5 FSC) was reconstructed as for Q23-huntingtin and shown in different angles rotated about the y axis (0°, 90°, 180°, 270°). (D) Sectioned view of 3D EM map of Q78-huntingtin in the same orientation as in C also showing a large cavity inside of Q78-huntingtin. This figure has additional supplement files: Figure supplement 1, 2, 3, 4, and 5 https://doi.org/10.7554/eLife.11184.005 We then attempted to locate the amino-terminus (17 residues adjacent to the polyglutamine tract) of huntingtin in the EM maps, by collecting images of negatively-stained purified complexes of antibody-bound amino-terminal FLAG-tags of the Q23- and Q78-huntingtin (Figure 3 and Figure 3—figure supplement 1 and 2). We also proceeded to reconstitute 3D structure of Q23- and Q78-huntingtin-antibody complexes. Comparisons of the 2D class averages and 3D reconstituted structures between huntingtin alone and the huntingtin-FLAG-antibody complex pairs clearly reveals an extra density at the top of the structure (in the view shown) for both Q23- and Q78-huntingtin (Figure 3). These observations strongly imply that the extreme N-terminus, and by inference the adjacent polyglutamine tract, is folded back, forming a spherically shaped solenoid with an internal cavity, but is accessible at the outside surface, regardless of its length. Figure 3 with 2 supplements see all Download asset Open asset Detection of the amino-terminus region of huntingtin by electron microscopy. 3D reconstitutions of Q23-huntingtin and Q23-huntingtin antibody complex (A) or Q78-huntingtin and Q78-huntingtin antibody complex (B) are shown in gray and yellow or in green and brown, respectively. 2D class averages corresponding to each huntingtin and its antibody complex are shown below the 3D reconstituted EM map and the extra density is marked with a red triangle. The extra-density indicating antibody on 3D EM map is colored in black (A) or dark brown (B) with red triangles. https://doi.org/10.7554/eLife.11184.011 Cross-linking-MS analysis reveals a modulated network of intramolecular contacts To further examine structural characteristics such as folding, we then assessed intramolecular interactions within huntingtin as estimated from the spatial proximity of lysine residues in sucrose-gradient-selected monomeric Q23- and Q78-huntingtin, determined by disuccinimidyl suberate (DSS) cross-linking mass spectrometry (DSS XL-MS) analysis (Leitner et al., 2014) (Figure 4—figure supplement 1, Supplementary file 1). Based upon the spacing between the DSS-cross-linked lysine residues in the primary sequence, the interactions that were detected for either huntingtin can be grouped into three categories, depicted in Figure 4A. These comprise: #1) short-range interactions (within a 200 amino acid interval), which seem likely to occur within the same secondary structure element, including the contacts between pairs of adjacent lysines (e.g. K174-K178 and K664-K669), and the interactions between K220, K255 and K262; #2) mid-range interactions (201 to 1000 amino acid interval), such as K826-K1559, K943-K1559 and K2548-K2934; and #3) long-range contacts (interval of >1000 amino acids), including between the carboxyl-terminal K2969 and amino-terminal K943 residue. Inspection of the depiction of the short-range cross-link contact sites relative to the location of the protease-sensitive sites (Seong et al., 2010) indicates that huntingtin is likely to be composed of five distinct domains (Figure 4A upper panel). The location of the protease-sensitive major hinge region located at residues 1184–1254 defines the 150 kDa amino-terminal domain (NTD) and the 200 kDa carboxyl-terminal domain (CTD). A minor-protease sensitive site located at ~ residue 500 demarcates the NTD into NTD-1 and NTD-II. Interestingly, a region centrally located within the CTD showed strikingly few crosslinks, despite the presence of several lysine residues, strongly implying a distinct sub-domain that we call ‘uncrosslinked’ sub-domain (UCD), which, given the paucity of short range intramolecular contacts, may adopt a largely unfolded structure. The UCD is flanked by regions with numerous intramolecular contacts; the CTD-I in proximity to the major proteolysis site and the carboxyl-terminal CTD-II (Figure 4A). Consistent with the hypothesis of five discernable huntingtin sub-domains, the results of hydrophobicity analysis show a transition in hydrophobicity prediction at the edge of each sub-domain (data not shown). Figure 4 with 1 supplement see all Download asset Open asset Cross-linking mass spectrometry analysis shows the intra-molecular interactions of Q23-, Q78- and Q46-huntingtin. The 3,144 amino acid primary huntingtin sequence (by convention Q23-huntingtin) is depicted as a yellow bar with the location of the polyglutamine tract indicated by the green arrowhead (A). The short-, mid- and long-range Lys-Lys cross-links by DSS identified in Q23-huntingtin (above the bar, Q23) and Q78-huntingtin (below the bar, Q78) by XL-MS are depicted by the green, blue and red-colored lines, respectively. Below that is a schematic view of huntingtin with five sub-domains delineated by the shared patterns of intra-molecular interactions; two amino-terminal (NTD-I, NTD-II) and three carboxyl-terminal (CTD-I, UCD and CTD-II), as defined relative to the landmark major protease-sensitive site at ~ residue 1200 identified previously (Seong et al., 2010), which is denoted by the large red arrowhead, while the secondary minor cleavage site is denoted by the small red arrowhead. The cross-links of Q46 huntingtin (Q46) identified in XL-MS analysis are also shown under the five sub-domains schematic. Lys-Lys cross-links by DSS unique to Q23-huntingtin, Q78-huntingtin and Q46-huntingtin are shown in cyan, pink, and orange, respectively in each pair-wise comparison of Q23 vs Q78 (B), Q23 vs Q46 (C) or Q46 vs Q78 (D). The amino-terminal (yellow) and carboxyl-terminal (blue) sub-domains are depicted in cartoons (E) to show more substantial interactions (red thicker dashed arrows) between NTD-I and CTD-I in Q23-huntingtin (left) and between CTD-II and NTD-II or CTD-I in Q78-huntingtin (right) and throughout both amino- and carboxyl-terminal sub-domains in Q46-huntingtin (middle). In all three huntingtins (all red dashed arrows), NTD-I folds to contact CTD-I and CTD-II also contacts CTD-I as well as NTD-II, implying that the physical impact of the polyglutamine tract at the very amino-terminal end has the opportunity to subtly but globally alter the entire huntingtin structure and function in a polyglutamine length-dependent manner. https://doi.org/10.7554/eLife.11184.014 The mid- and long-range interactions occur between these sub-domains in a pattern that indicates close-proximity of the extreme amino- and carboxyl-terminal sub-domains. Specifically, NTD-I mainly interacts with CTD-I, while CTD-II interacts with NTD-II and notably with CTD-1. Thus, the pattern of mid- and long-range contacts supports a view of huntingtin folding such that the extreme amino-terminal subdomain (NTD-I), with its polyglutamine tract, and the extreme carboxyl-terminal subdomain (CTD-II) are close to each other by virtue of contacts that each makes with the NTD-II and CTD-I sub-domains that flank the major cleavage site. Notably, the overall contact-patterns for the Q23- and Q78-huntingtin were similar, supporting observations of a generally similar core-stability (Figure 1) and shape (Figures 2 and 3). However, subtraction of the 38 crosslinks common to both proteins highlight networks of contacts that are relatively specific for either Q23-huntingtin (13 crosslinks) or Q78-huntingtin (8 crosslinks), as depicted in Figure 4B. To further examine the patterns of the internal interaction depending on its polyglutamine length, we also performed XL-MS analysis of Q46-huntingtin. First, the overall contact-patterns of Q46-huntingtin were similar and consistent with the five distinct domains (Figure 4A lower panel). Compared with those of Q23- and Q78-huntingtin, the unique contacts of Q46 reveal widespread interactions across the entire region of the protein (Figure 4C and D). These unique, largely mid- and long-range contacts disclose that Q23-huntingtin exhibits more unique interactions of the NTD-I with the CTD-I, and Q78-huntingtin displays more unique contacts between the CTD-II and the CTD-I and on occasion with NTD-II. On the other hand, the unique crosslinks of Q46-huntingtin reveal that CTD-I seems to interact with both NTD-I and CTD-II as if Q46-huntingtin posits an intermediate conformation between Q23- and Q78-huntingtin. (Figure 4E). These observations imply a subtle but detectable 3-dimensional structural impact of polyglutamine tract length as it increases. Discussion We applied a systematic structure-function approach to delineate the features of huntingtin that conspire with its polyglutamine tract to comprise, in conjunction with some as yet unknown target, the dominant gain of function mechanism that triggers the pathogenic process in patients with HD. Our biophysical analyses of an allelic series of native recombinant human huntingtins now provide a satisfying solution to the mystery of how the amino-terminal polyglutamine tract may be in a position to modulate huntingtin structure and function. The results of EM and XL-MS analyses provide coherent support for a HEAT/HEAT-like repeat solenoid comprising a major hinge that delimits two large nearly equal-sized domains that fold such that the ends of each arm are in close proximity and the whole circumscribes an extensive internal cavity. Other HEAT repeat proteins such as nuclear importin and exportins have functional protein-protein binding interfaces located at the inner side of the solenoid structure (Chook and Blobel, 2001; Cingolani et al., 1999). Considering that the size of huntingtin is much bigger than other HEAT repeat proteins, we can imagine that the HEAT repeat domains can be folded back to form a closed structure that we have observed in huntingtin, having functional sites located in the internal cavity. This shape classifies huntingtin as a closed helical solenoid, contrasting with the shorter open curvilinear HEAT/HEAT-like repeat solenoids whose native structures have been solved at high resolution (Cingolani et al., 1999; Groves et al., 1999). Huntingtin’s distinctive shape is predicted to provide both internal and external surface topologies that may mediate the binding of proteins or nucleic acids, as befitting a mechanical HEAT/HEAT-like repeat interaction-scaffold (Takano and Gusella, 2002). Our biophysical analyses also provide basic insights for future higher-resolution studies that will be needed to more precisely delineate huntingtin structure and its modulation by the polyglutamine tract. The DSS-XL-MS intramolecular cross-linking data, together with our previously reported limited proteolysis analysis (Seong et al., 2010) provide a general sense of how rod-like α-helical HEAT/HEAT-like repeat domains, may fold to delimit the closed shape that we observe. The pattern of proteolysis, regardless of polyglutamine tract length, revealed a single major cleavage-sensitive site at ~ residue 1200, strongly predicting a major hinge or pivot that roughly parses the protein into two nearly equal ‘arms’, a 150 kDa NTD and a 200 kDa CTD. This is supported by the patterns of short- and mid-range intramolecular DSS-XL-MS delineated intramolecular contacts that are shared by the Q23-, Q46- and Q78-huntingtin, which are mainly located within and between each of the regions that immediately flank this location, implying extensive local internal folding and close proximity of the ‘arms’ around the pivot-point. Multiple short- and mid-range interactions are also detected at the ends of the NTD-I and CTD-II. These contacts imply internal folding near the ends of each arm. Limited proteolysis of the amino-terminal domain did reveal an internal cleavage site, located at ~ residue 500, which is consistent with internal pivot points, along with other internal folding, that may explain the short- and mid-range contacts detected by XL-MS near the terminus. However, the carboxyl-terminal domain lacked internal cleavage-accessible sites (Seong et al., 2010), implying a paucity of accessible hinge-points. Consistent with this, XL-MS fails to detect short- or mid-range cross-linked lysine residues in the sub-region spanning amino acids ~1800 to ~2300, except one long distance contact and one short distance contact only in Q46-huntingtin. This sub-region contains 13 lysine residues. It is possible that these residues are not DSS-accessible although we did observe DSS modified, but not cross-linked peptides in this region (data not shown), implying instead a more extended 3D structure that maximizes the surface area available for interaction with binding-partners, as in other HEAT repeat solenoid proteins (Cingolani et al., 1999; Grinthal et al., 2010; Groves et al., 1999). By contrast, the adjacent extreme C-terminus does display some internal short- and mid-range cross-linking interactions that suggest internal folding, though apparently without a well-defined proteolysis-accessible hinge-point. However, perhaps the most striking finding is the multiple long-range interaction cross-links between the CTD-II and CTD-I or NTD-II, as well as the long-range contacts of the NTD-I with the CTD-I close to the major hinge-pivot, which places the ends of each arm in close proximity to each other on the carboxyl-terminal domain arm, above the major hinge. Of note, EM analysis of the amino-terminal FLAG-antibody-huntingtin complex strongly suggests accessibility of the extreme N-terminus, and likely the adjacent polyglutamine tract, at the external surface. Thus, folding of the two main HEAT/HEAT-like domains forms an extensive internal cavity consistent with the shape that we observe in EM analysis, while providing an elegant explanation for the conundrum of how the polyglutamine tract located at the end of the amino-terminal arm may affect change throughout the entire protein. All of the huntingtins, regardless of polyglutamine tract length, appear to have the same basic core structure. Our CD data demonstrates that they are all α-helical and have the same pattern of thermal stability, denaturing over the same temperature range. They have similar shapes in EM analysis and display mostly shared DSS-XL-MS intramolecular interactions, as discussed above. Nevertheless the differences among huntingtins with different polyglutamine lengths (Q23, Q46 and Q78) are telling. Each displays a unique intramol

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat alpha-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes. Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large alpha-helical domains. Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9. Supporting a direct stimulatory role, full-length recombinant huntingtin significantly increased the histone H3K27 tri-methylase activity of reconstituted PRC2 in vitro, and structure-function analysis demonstrated that the polyglutamine region augmented full-length huntingtin PRC2 stimulation, both in Hdh(Q111) EBs and in vitro, with reconstituted PRC2. Knowledge of full-length huntingtin's alpha-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region.

Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated Hdh(Q111) knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four Hdh(Q111) phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.