Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription
- University of Texas Medical Branch, United States
- University of California, Irvine, United States
- Duke University School of Medicine, United States
- Sam Houston State University, United States
- Houston Methodist Research Institute, United States
- Buck Institute for Research on Aging, United States
- University of California, Irvine, Institute for Memory Impairments and Neurological Disorders, United States
Figures
Figure 1 with 7 supplements
HTT is a part of the TCR complex.
(A) Nuclear extract (NE), and cytosolic extract (CE) were purified from human neuroblastoma SH-SY5Y cells and the protein fractions were analyzed by western blots (WBs) to detect HTT, ATXN3, PNKP, …
Figure 1—figure supplement 1
Huntingtin is a part of the transcription-coupled DNA repair (TCR) complex.
Nuclear extracts (NEs) from human neuroblastoma SH-SY5Y cells was isolated and the endogenous HTT was immunoprecipitated (IP’d) with anti-HTT antibody. The HTT immunocomplex (HTT-IC) was analyzed by …
Figure 1—figure supplement 2
ATXN3 is present in the HTT-TCR complex.
Nuclear extracts (NEs) from human neuroblastoma SH-SY5Y cells was isolated and the endogenous ataxin-3 was immunoprecipitated (IP’d) with anti-ataxin-3 antibody. The atxin-3 immunocomplex (ATXN3-IC) …
Figure 1—figure supplement 3
DNA strand break repair enzyme PNKP is present in the TCR complex.
Endogenous PNKP was IP’d from NEs of SH-SY5Y cells and PNKP IC was analyzed by WBs to examine the presence of PNKP-associated TCR components. The original WBs show the protein bands and molecular …
Figure 1—figure supplement 4
RNA polymerase large subunit (POLR2A) is present in the HTT-TCR complex.
POLR2A was IP’d from NEs of SH-SY5Y cells and IC was analyzed by WBs to detect the POLR2A-associated TCR proteins. Presence of HAP-1, ATXN3, PNKP, POLR2A, LIG 3, CBP, and TAFII 130 (TAF4) are shown …
Figure 1—figure supplement 5
Wild-type normal HTT is a part of the multiprotein TCR complex in vitro.
Nuclear extract (NEs) was isolated from PC12 cells exogenously expressing Myc-tagged full-length wild-type normal HTT encoding 23Qs (FL-HTT-Q23), and the Myc-HTT-Q23 protein was immunoprecipitated …
Figure 1—figure supplement 6
Mutant HTT is a part of the multiprotein TCR complex in vitro.
Nuclear extract (NEs) was isolated from PC12 cells ectopically expressing a Myc-tagged full-length mutant HTT encoding 148Qs (FL-mHTT-Q148) for assessing the possible interaction of mutant HTT with …
Figure 1—figure supplement 7
HTT is a component of the TCR complex in vivo.
(A) Proximity Ligation Analysis (PLA) was performed on normal (left) and HD (expressing mHTT-Q58, adult onset HD patient, disease grade 4/4 and manifested severe phenotypes) patient brain sections …
Figure 2
HTT colocalizes with PNKP and ATXN3 in postmortem human brain sections.
(A) Normal and HD postmortem brain (mHTT-Q94) sections were analyzed by double immunolabeling with antibodies against HTT (green) and PNKP (red) to assess their in vivo colocalization and possible …
Figure 3
HTT interacts with the C-terminal catalytic domain of PNKP.
(A) Schematic illustrating various functional domains of PNKP expressed as FLAG-tagged peptides: (1) full-length PNKP containing N-terminal fork-head-associated (FHA) domain, central phosphatase …
Figure 4
The N-terminus of HTT interacts with the C-terminal catalytic domain of PNKP.
(A) Schematic showing GFP-tagged N-terminal fragment of wild-type normal HTT encoding 23Qs or mutant HTT encoding 97Qs (NT-wtHTT-Q23 and NT-mHTT-Q97 plasmid vectors respectively; upper panel). …
Figure 5 with 5 supplements
mHTT abrogates PNKP activity in vitro and in vivo.
(A) The 3'-phosphatase activities of PNKP in the NE (250 ng each) of control (lanes 1 and 2, differentiation replicates of Q33 iPSCs), and HD neurons (lanes 3 and 4, differentiation replicates of …
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Figure 5—source data 1
Huntinton's disease models.
- https://doi.org/10.7554/eLife.42988.020
Figure 5—figure supplement 1
Endogenous level of mHTT is sufficient to deplete nuclear PNKP activity in iPSC-derived HD primary striatal neurons.
(A) The 3'-phosphatase activities of PNKP in the NE (250 ng each) of control (lanes 2, 3 and 4; differentiated iPSCs expressing Q18, Q28 and Q33), and HD neurons (lanes 5, 6 and 7; differentiated …
Figure 5—figure supplement 2
mHTT-mediated inactivation of the TCR complex abrogates PNKP activity.
(A) The 3'-phosphatase activities of PNKP in the NE (250 ng each) of WT (Cntl: lane 2), wtHTT-Q23-expressing (lane 3), and mHTT-Q148-expressing (lane 4) PC12 cells were determined by measuring …
Figure 5—figure supplement 3
Expression of the N-terminus of mHTT abrogates PNKP activity.
(A) NEs were isolated from WT PC12 cells (Cntl; lane 2) or cells expressing NT-wtHTT-Q23 (lane 3) or NT-mHTT-Q148 (lane 4), and PNKP activity was assessed. (B) Relative amounts of phosphate release …
Figure 5—figure supplement 4
mHTT triggers DNA damage response (DDR)-ATM signaling.
(A) Total protein from the induced pluripotent stem cell (iPSC)-derived primary control neurons (Q18 and Q28) and HD primary neurons (Q50 and Q53) was isolated and analyzed by WBs to detect …
Figure 5—figure supplement 5
PNKP overexpression in mutant cells rescues cell toxicity.
(A) NEs isolated from the mutant SH-SY5Y cells (NT-mHTT-Q97) (lane 2) and mutant cells with PNKP overexpression (lane 3), and PNKP activity was measured. No protein was added in lane 1 (NP) and …
Figure 6 with 4 supplements
mHTT preferentially induces DNA damage/strand breaks in the transcriptionally active genome.
(A) Tissue from CTX and STR of 5 WT mouse brain (7 weeks old) was pooled and total RNA was isolated and expression levels of various genes were measured using qRT-PCR analysis. Left panel shows …
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Figure 6—source data 1
Mutant Huntingtin- mediated CBP degradation.
- https://doi.org/10.7554/eLife.42988.026
Figure 6—figure supplement 1
mHTT expression induces DNA strand breaks in STR but not in CRBL.
(A) Genomic DNA was isolated from the STR of asymptomatic zQ175 transgenic (STR from three transgenic mice were pooled) and age-matched WT control (STR from three control mice were pooled) mice. …
Figure 6—figure supplement 2
HD patients’ brain and HD transgenic mouse brain accumulate DNA damages.
(A) A representative confocal image showing immunostaining of HD patients’ brain section expressing mHTT-Q58 (lower panel) and age-matched normal control brain section (upper panel) with …
Figure 6—figure supplement 3
HD primary neurons accumulate DNA breaks preferentially in the actively transcribing genome.
(A) Control and HD primary neurons expressing Q18 and Q50 respectively were immunostained with anti-γH2AX antibody to detect DNA strand breaks (arrows). (B) Control and HD primary neurons expressing …
Figure 6—figure supplement 4
N171-82Q transgenic mouse brain predominantly accumulates strand breaks in the transcriptionally active genome.
LA-qPCR analysis. Genomic DNA was isolated from the CTX of symptomatic (16 wks old) N171-82Q transgenic (n = 3, pooled) and age-matched control mice (n = 3, pooled), various genomic loci were PCR …
Figure 7
mHTT facilitates CBP degradation by inactivating ATXN3.
(A) Nuclear extracts (NEs) isolated from control primary neurons (Q18 and Q28) and HD neurons (Q53 and Q109) were analyzed by WBs to measure PNKP, ATXN3, POLR2A, CBP, and CREB levels; HDAC2 was the …
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Figure 7—source data 1
DNA damage precedes neurodegeneration.
- https://doi.org/10.7554/eLife.42988.028
Figure 8
Proposed mechanism by which mHTT triggers neurotoxicity in HD.
Schematic diagram of our hypothesized mechanism by which polyQ expansions in mHTT compromise the functional integrity of the TCR complex. Normal HTT forms a multiprotein TCR complex with POLR2A, …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.42988.030
