Histone deacetylase knockouts modify transcription, CAG instability and nuclear pathology in Huntington disease mice
- Center for Genomic Medicine, Harvard Medical School, United States
- Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, United States
- Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, United States
- Department of Neurology, Massachusetts General Hospital, Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
MSN-specific deletion of Hdac2 or Hdac3.
(A) D9-Cre mice, expressing Cre recombinase from the Ppp1r1b promoter from 5 to 6 weeks of age, were crossed with floxed Hdac2 or Hdac3 mice (schematic illustrates exons flanked by loxP sites), and …
Figure 1—figure supplement 1
Detailed mating scheme.
(A) The scheme is shown for Hdac2 but the same scheme was implemented for Hdac3. Mice carrying conditional floxed Hdac2 or Hdac3 alleles were crossed with HttQ111 mice (cross 1) and, in a separate …
Figure 1—figure supplement 2
Upregulation of HDAC1 in Hdac2 KO striata.
(A) Total lysates of contralateral striata of a subset of 5-month Hdac2 KO mice from the RNAseq cohort (N = 4 for each of the four compared genotypes, as indicated on the bar graphs), probed with …
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Figure 1—figure supplement 2—source data 1
HDAC2 and HDAC1 protein levels in Hdac2 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Hdac2 or Hdac3 knockout in striatal MSNs does not affect HDAC2 or HDAC3 levels in cortex.
Shown are fluorescent micrographs of somatosensory cortex of 10-week-old Hdac2 KO (A) and Hdac3 KO (B) mice immunostained with anti-HDAC2 and anti-HDAC3 antibody, respectively.
Figure 2 with 4 supplements
RNA-seq analyses in HttQ111 striata with MSN-specific Hdac2 knockout.
Results of differential gene expression analyses showing the number of differentially expressed genes (DEGs) for each of five contrasts. Number and % of DEGs at nominal p<0.05 or FDR adjusted p<0.05 …
Figure 2—figure supplement 1
Expression of Hdac genes and Cre recombinase.
(A) Cre expression is increased in Hdac2 KO mice that also express the DARPP-32-Cre transgene. (B) Expression level of each Hdac2 exon showing reduced expression of exons 2–4 in Hdac2 KO mice, as …
Figure 2—figure supplement 2
Principal Components Analyses (PCA).
PC1 separates gene expression by Hdac2 genotype (A), while PC2 separates gene expression largely by sex (B). Gene expression data does not clearly separate the samples by HttQ111 genotype (C).
Figure 2—figure supplement 3
Global H3K9 and H4K12 acetylation levels.
(A) Quantification of acetylated Lys9 on histone H3, normalized by total H3 level, as detected by western blot. ‘Relative units’ represent the density of AcH3K9 band divided by the density of the …
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Figure 2—figure supplement 3—source data 1
AcH3K9 and AcH4K12 levels in Hdac2 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig2-figsupp3-data1-v2.xlsx
Figure 2—figure supplement 4
Cross-study comparison of HttQ111-differentially expressed genes.
RNA-seq data from striata of B6J.HttQ111/+ and Htt+/+ mice described in Langfelder et al., 2016 were analyzed in the same way as the current study for comparison. (A) The number of dysregulated …
Figure 3 with 1 supplement
Pathway enrichment of differentially expressed genes.
The most significantly enriched pathways that are up- or down-regulated in each of four differential gene expression contrasts are shown. Numbers at the end of each bar show the number of genes …
Figure 3—figure supplement 1
Overlaps of differentially expressed genes and enriched pathways in each of the four contrasts.
Numbers of DEGs genes (A) and enriched pathways (B) that overlap between the four contrasts at different significance threshold cut-offs. (C) The significance, determined using a one-tailed Fisher …
Figure 4 with 1 supplement
Rescue by Hdac2 KO of gene expression levels of a subset of genes dysregulated by the HttQ111 allele.
(A) To identify genes dysregulated by the HttQ111 allele and by Hdac2 KO in HttQ111/+ mice, the overlapping genes in contrasts 3 and 2 were identified (left table). This shows the numbers of genes …
Figure 4—figure supplement 1
Impact of Hdac2 KO on the most significantly dysregulated genes common to this study and Langfelder et al.
Box-plots showing expression levels of the 29 genes significantly dysregulated in HttQ111/+ striata (FDR-adjusted p<0.05) in both the current study and in Langfelder et al., 2016 across all four …
Figure 5
Hdac2 KO reduces the relative impact of the HttQ111 allele.
Absolute log2 fold-change of gene expression differences between HttQ111/+ and Htt+/+ were determined in Hdac2 WT mice and in Hdac2 KO mice as a measure of gene expression change regardless of …
Figure 6 with 3 supplements
Deletion of Hdac2 or Hdac3 in striatal MSNs decreases striatal HTT CAG repeat expansions.
(A) Representative GeneMapper traces of PCR-amplified striatal HTT CAG repeats from individual mice matched by main allele repeat length within each age group (Source data 1). Main allele is marked …
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Figure 6—source data 1
Expansion Indices in Hdac2 and Hdac3 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Expression of Cre transgene does not affect HTT CAG repeat instability and nuclear huntingtin accumulation in MSNs.
CAG instability and nuclear mutant huntingtin were measured in a cohort of 5 month HttQ111/+ mice harboring wild-type Hdac2 and Hdac3 alleles, either with or without the DARPP-32 Cre transgene (D9-Cr…
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Figure 6—figure supplement 1—source data 1
Expansion Indices and mAb5374 nuclear huntingtin immunostaining intensities in Cre-expressing HttQ111 mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig6-figsupp1-data1-v2.xlsx
Figure 6—figure supplement 2
Effect of Hdac2 or Hdac3 deletion in striatal MSNs on number of DARPP-32-positive cells and DARPP-32 levels.
DARPP-32 levels (mean integrated intensity; right panels) are quantified as integrated intensity of DARPP-32 immunostaining, normalized by the number of DARPP-32-positive cells (left panels). Refer …
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Figure 6—figure supplement 2—source data 1
DARPP32-positive cells and DARP32 levels in Hdac2 and Hdac3 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig6-figsupp2-data1-v2.xlsx
Figure 6—figure supplement 3
DNA repair gene expression.
(A) Differential expression analysis of DNA repair genes from RNA-seq data in 5-month mice. (B) Effect of Hdac2 KO on levels of Msh3 mRNA from RNA-seq data in 5-month mice. (C) Total lysates of …
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Figure 6—figure supplement 3—source data 1
MSH3 protein levels in Hdac2 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig6-figsupp3-data1-v2.xlsx
Figure 7
HDAC inhibitors increase instability in selectable cell-based assays for CAG repeat contraction.
(A) HPRT mini-gene reporter construct for contraction assay. (B) Fold-change in the number of HAT-resistant FLAH25 cells (HPRT+) when treated with HDAC inhibitors relative inhibitors relative to the …
Figure 8 with 1 supplement
Impact of MSN-specific Hdac2 or Hdac3 knockout on striatal nuclear mutant huntingtin immunostaining phenotypes.
(A) Fluorescent micrographs of HttQ111/+ Hdac2 KO striata immunostained with anti-huntingtin mAb5374 and histone H3 antibodies. Mice were matched by CAG repeat length within each age group. Images …
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Figure 8—source data 1
mAb5474 nulcear intensity and number of nuclear inclusions in Hdac2 and Hdaac3 KO mice.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Huntingtin expression.
(A) Effect of Hdac2 KO on levels of total huntingtin mRNA (A) and on aberrant huntingtin transcripts spanning exon 1 + intron 1 (B) from RNA-seq data in 5-month mice. In panel B, reads were aligned …
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Figure 8—figure supplement 1—source data 1
Zip file: RNAseq_codes_and_files.
- https://cdn.elifesciences.org/articles/55911/elife-55911-fig8-figsupp1-data1-v2.xlsx
Additional files
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Source code 1
RNA-seq codes and data files.
- https://cdn.elifesciences.org/articles/55911/elife-55911-code1-v2.zip
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Source data 1
CAG lengths of mouse cohorts.
- https://cdn.elifesciences.org/articles/55911/elife-55911-data1-v2.docx
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Source data 2
Differentially expressed genes.
- https://cdn.elifesciences.org/articles/55911/elife-55911-data2-v2.xlsx
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Source data 3
Pathway analyses.
- https://cdn.elifesciences.org/articles/55911/elife-55911-data3-v2.xlsx
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Source data 4
Rescue analysis.
- https://cdn.elifesciences.org/articles/55911/elife-55911-data4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55911/elife-55911-transrepform-v2.docx
