The Journal of Neuroscience, March 8, 2006, ():
Differential Contributions of Caenorhabditis elegans Histone Deacetylases to Huntingtin Polyglutamine Toxicity
J. Neurosci. Bates et al.
26: 2830
Supplemental data
Files in this Data Supplement:
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Supplemental Table 1: Double stranded RNA was expressed by cloning fragments of target genes into the L4440 feeding vector. Gene targets, primer sequences, and restriction enzymes used for the cloning are listed.
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Supplemental Table 2: HDAC gene function was reduced by feeding dsRNA. HDAC gene name is listed in column 1. Reducing function of most HDACs increased ASH neuron degeneration. hda-4 (C10E2.3 was previously published as hda-7) function was reduced using a loss of function allele. Note that C. elegans has no hda-7, hda-8, or hda-9 genes. Only reducing function of hda-3 decreased degeneration. Standard deviation and n are listed in columns 4 and 5 respectively.
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Supplemental Figure 1: Chemical inhibition of class I and class II HDACs: Htn-Q150-induced degeneration is reduced from 62+/-9% to 26+/-3% by 150 ug/ml TSA in 8 day old rtIs11Htn-Q150 animals. Results are a compilation of 2 trials totaling over 100 neurons. Error bars represent the standard deviation. Two-tailed T-Test analysis yields p<0.06 for each comparison.
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Supplemental Figure 2: Phylogeny of HDACs: C. elegans HDACs and representative human and yeast HDACs compared using ClustalW hda-1 (C53A5.3), hda-2 (C08B11.2), hda-3 (R06C1.1), hda-4 (C10E2.3), hda-5 (F43G6.11), hda-6 (F41H10.6), hda-10 (Y51H1A.5), hda-11 (C35A5.9), sir-2.1 (R11A8.4), sir-2.2 (F46G10.7), sir-2.3 (F46G10.3) are C. elegans HDACs. Representative Homo sapiens HDACs include HDAC1, RPD3, HDAC2, HDAC3, HDAC10, HDAC6, and Sir-2 like. Representative Saccharomyces HDACs are HDA1 and Sir2.
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Supplemental Figure 3: Ce-hda-3 is ubiquitously expressed and localizes to the nucleus in the ASH neurons. A. Ce-hda-3 protein in nuclei of C. elegans cells was visualized using Ce-hda-3 polyclonal antibody staining. B. OSM-10 antisera was used to identify an ASH neuron. The arrow points to the same ASH neuron in both images. Bar equals 10μm.
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Supplemental Figure 4: hda-1(RNAi) and hda-3(RNAi) do not modulate necrotic cell death.
A. deg-1(rt70) animals were raised at 20°C resulting in low levels of ASH neuron necrotic degeneration. Neither hda-1(RNAi) nor hda-3(RNAi) enhanced degeneration. Two-tailed T-test pair-wise comparisons with controls yield p>0.4. B. deg-1(rt70) animals were raised at the restrictive temperature, 15°C resulting in high levels of necrotic ASH neuron degeneration. Neither hda-1(RNAi) nor hda-3(RNAi) suppressed degeneration. Error bars represent standard deviation. Two-tailed T-test pair-wise comparisons with controls yield p>0.3.
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Supplemental Figure 5: Transgene silencing does not contribute to HDAC modulation of ASH neurodegeneration. A. ASH neuron degeneration is quantified in rtIs11Htn-Q150 animals, rrf-3;rtIs11Htn-Q150 animals, and ncl-1;rtIs11Htn-Q150 animals. Roles for ncl-1 and rrf-3 in transgene silencing were previously described (Kim et al., 2005), but they do not alter Htn-Q150 toxicity in ASH neurons. Controls are combined. Error bars represent standard deviation. Two-tailed T-test pair-wise comparisons with controls yield p>0.35. B. Htn-Q150 transgene levels do not significantly change in 3 day old rtIs18Htn-Q150 animals when hda-3 or hda-1 are reduced by RNAi. Htn-Q150 transgene levels were measured using quantitative PCR and compared to osm-6 mRNA levels as a control for the total quantity of RNA loaded. This graph represents data from two trials that were averaged together. Two-tailed T-test pair-wise comparisons with controls yield p>0.85. Degeneration of ASH neurons in sibling 8 day old animals was confirmed to average 44+/-7% in control animals, 17+/-6% in hda-3(RNAi);rtIs18Htn-Q150, and 67+/-6% in hda-1(RNAi);rtIs18Htn-Q150 animals. Two-tailed pairwise T-test yields p<.05.
