A Systematic Review of Transcriptional Dysregulation in Huntington’s Disease Studied by RNA Sequencing

Huntington's disease (HD) is a chronic neurodegenerative disorder caused by an expansion of polyglutamine repeats in exon 1 of the Huntingtin gene. Transcriptional dysregulation accompanied by epigenetic alterations is an early and central disease mechanism in HD yet, the exact mechanisms and regulators, and their associated gene expression programs remain incompletely understood. This systematic review investigates genome-wide transcriptional studies that were conducted using RNA sequencing (RNA-seq) technology in HD patients and models. The review protocol was registered at the Open Science Framework (OSF). The biomedical literature and gene expression databases, PubMed and NCBI BioProject, Array Express, European Nucleotide Archive (ENA), European Genome-Phenome Archive (EGA), respectively, were searched using the defined terms specified in the protocol following the PRISMA guidelines. We conducted a complete literature and database search to retrieve all RNA-seq-based gene expression studies in HD published until August 2020, retrieving 288 articles and 237 datasets from PubMed and the databases, respectively. A total of 27 studies meeting the eligibility criteria were included in this review. Collectively, comparative analysis of the datasets revealed frequent genes that are consistently dysregulated in HD. In postmortem brains from HD patients, DNAJB1, HSPA1B and HSPB1 genes were commonly upregulated across all brain regions and cell types except for medium spiny neurons (MSNs) at symptomatic disease stage, and HSPH1 and SAT1 genes were altered in expression in all symptomatic brain datasets, indicating early and sustained changes in the expression of genes related to heat shock response as well as response to misfolded proteins. Specifically in indirect pathway medium spiny neurons (iMSNs), mitochondria related genes were among the top uniquely dysregulated genes. Interestingly, blood from HD patients showed commonly differentially expressed genes with a number of brain regions and cells, with the highest number of overlapping genes with MSNs and BA9 region at symptomatic stage. We also found the differential expression and predicted altered activity of a set of transcription factors and epigenetic regulators, including BCL6, EGR1, FOSL2 and CREBBP, HDAC1, KDM4C, respectively, which may underlie the observed transcriptional changes in HD. Altogether, our work provides a complete overview of the transcriptional studies in HD, and by data synthesis, reveals a number of common and unique gene expression and regulatory changes across different cell and tissue types in HD. These changes could elucidate new insights into molecular mechanisms of differential vulnerability in HD. Systematic Review Registration: https://osf.io/pm3w

A novel mechanism for the establishment of sister chromatid cohesion by the ECO1 acetyltransferase

Cohesin complex mediates cohesion between sister chromatids, which promotes high-fidelity chromosome segregation. Eco1p acetylates the cohesin subunit Smc3p during S phase to establish cohesion. The current model posits that this Eco1p-mediated acetylation promotes establishment by abrogating the ability of Wpl1p to destabilize cohesin binding to chromosomes. Here we present data from budding yeast that is incompatible with this Wpl1p-centric model. Two independent in vivo assays show that a wpl1∆ fails to suppress cohesion defects of eco1∆ cells. Moreover, a wpl1∆ also fails to suppress cohesion defects engendered by blocking just the essential Eco1p acetylation sites on Smc3p (K112, K113). Thus removing WPL1 inhibition is insufficient for generating cohesion without ECO1 activity. To elucidate how ECO1 promotes cohesion, we conducted a genetic screen and identified a cohesion activator mutation in the SMC3 head domain (D1189H). Smc3-D1189H partially restores cohesion in eco1∆ wpl1∆ or eco1 mutant cells but robustly restores cohesion in cells blocked for Smc3p K112 K113 acetylation. These data support two important conclusions. First, acetylation of the K112 K113 region by Eco1p promotes cohesion establishment by altering Smc3p head function independent of its ability to antagonize Wpl1p. Second, Eco1p targets other than Smc3p K112 K113 are necessary for efficient establishment.National Institutes of Health (U.S.) (Grant R01GM092813

Ten simple rules for implementing open and reproducible research practices after attending a training course

Open, reproducible, and replicable research practices are a fundamental part of science. Training is often organized on a grassroots level, offered by early career researchers, for early career researchers. Buffet style courses that cover many topics can inspire participants to try new things; however, they can also be overwhelming. Participants who want to implement new practices may not know where to start once they return to their research team. We describe ten simple rules to guide participants of relevant training courses in implementing robust research practices in their own projects, once they return to their research group. This includes (1) prioritizing and planning which practices to implement, which involves obtaining support and convincing others involved in the research project of the added value of implementing new practices; (2) managing problems that arise during implementation; and (3) making reproducible research and open science practices an integral part of a future research career. We also outline strategies that course organizers can use to prepare participants for implementation and support them during this process

Structural studies on the LINC complex and Fic-1

Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis.Includes bibliographical references (pages 37-38).LINC complexes span the nuclear envelope and connect the nucleoskeleton to the cytoskeleton. In 2012, our lab solved the first LINC complex structure, that of SUN domain of human SUN2 bound with KASH1 or KASH2 peptides. In this project testes-specific human SUN proteins (SUN3, SPAG4, and SUNS) were compared to ubiquitously-expressed SUN2. Secondly, fission and budding yeast LINC complexes differ from human ones and were analyzed as well. I was able to confirm SUN-KASH interaction in human and yeast. For structural analysis I explored various expression strategies. Fic-1 is a C. elegans Fic-domain protein with diverse cellular functions. As a subfamily III Fic enzyme, Fic-1 may reveal valuable insights into Fic enzyme mechanisms from its structure. After trying different knowledge-informed constructs and crystal optimization, small Fic-1 crystals were obtained, which diffracted X-rays to ~ 7 [angstroms]. With modest additional effort diffraction-quality crystals should be achievable.by Xuanzong Guo.S.M

The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.National Institutes of Health (U.S.) (P41 GM103403)Swiss National Science Foundation (Advanced Postdoc Mobility Fellowship)National Institutes of Health (U.S.) (NIH Pioneer Award (DP01)

The <i>Caenorhabditis elegans</i> Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones

<div><p>Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the <i>Caenorhabditis elegans</i> Fic protein FIC-1 <i>in vitro</i> and <i>in vivo</i>. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen <i>Pseudomonas aeruginosa</i>, establishing a connection between AMPylation and innate immunity in <i>C</i>. <i>elegans</i>.</p></div

Hyper- or hypo-AMPylation has no apparent consequences on nematode viability and response to acute or chronic ER stress.

<p>(A) AMPylation has no influence on development under acute ER stress: eggs were transferred to OP50 plates containing different concentrations of tunicamycin to induce acute ER stress. Embryo development was scored. Average of three independent experiments shown here. (B) AMPylation has no influence on development under chronic ER stress: eggs were transferred to <i>P</i>. <i>aeruginosa</i> plates to induce chronic ER stress. Embryo development was scored. Average of three independent experiments shown here. (C) and (D) development assay under chronic ER stress: eggs of indicated lines were transferred to <i>P</i>. <i>aeruginosa</i> plates to induce chronic ER stress. Embryo development was scored. Average of three independent experiments shown here.</p

FIC-1 is an AMPylase.

<p>(A) FIC-1 exhibits auto-AMPylation activity: Recombinant FIC-1, FIC-1 E274G or FIC-1 H404A was incubated with α ³³P-ATP for an hour and incorporation of label was assessed by SDS-PAGE and autoradiography. (B) FIC-1 accepts different nucleotide substrates: FIC-1 E274G or HYPE E234G were incubated with respective α ³³P-labeled nucleotides for one hour at room temperature and sample autoradiography was assessed. (C) FIC-1 E274G AMPylates histone H3: Recombinant FIC-1, FIC-1 E274G or FIC-1 H404A was incubated with α ³³P-ATP for an hour at which point histone H3 was added and the mixture was incubated for an additional hour. Incorporation of label was assessed by SDS-PAGE and autoradiography. (D) FIC-1 E274G/T476A and FIC-1 E274G/T352A are fully active: Recombinant FIC-1 E274G, FIC-1 E274G/T476A and FIC-1 E274G/T352A was incubated with α ³³P-ATP for an hour at which point histone H3 was added and the mixture was incubated for an additional hour. Incorporation of label was assessed by SDS-PAGE and autoradiography. (E) FIC-1 AMPylates core histones H2 and H3 but not H4: Recombinant FIC-1, FIC-1 E274G or FIC-1 H404A was incubated with α ³³P-ATP for an hour at which point purified histone substrates were added and the mixture was incubated for an additional hour. Incorporation of label was assessed by SDS-PAGE and autoradiography.</p

FIC-1 AMPylates conserved heat shock 70 family proteins and translation elongation factors.

<p>(A) Identification of new FIC-1 targets by mass spectrometry. (B) Validation of novel FIC-1 targets: Recombinant FIC-1 E274G was incubated with α ³³P-ATP for an hour at which point substrates (histone H3, HSP-1 or eEF-1A2) were added and the mixture was incubated for an additional hour. Sample autoradiography was assessed. (C) Novel FIC-1 targets are modified by HYPE: Recombinant HYPE E234G was incubated with α ³³P-ATP for an hour at which point substrates (HSP-1 or eEF-1A) were added and the mixture was incubated for an additional hour. Sample autoradiography was assessed.</p