A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, Janelia Research Campus, United States
Figures
Figure 1 with 1 supplement
The ubiquitin-proteasome system regulates TBP protein levels.
(A) Treatment of differentiated C2C12 myotubes with the proteasome inhibitor MG132 rescues TBP protein levels. Western blot analysis of cytoplasmic and nuclear extracts from C2C12 myoblasts and …
Figure 1—figure supplement 1
Protein levels of TBP and TAF4 but not RNA polymerase II significantly decrease during C2C12 differentiation.
Western blot analysis of whole-cell extracts prepared from C2C12 myoblasts (D0) and myotubes collected on day 3 (D3) and day 6 (D6) post-differentiation was done using antibodies against TAF4, TBP, …
Figure 2 with 1 supplement
Purification of TBP E3 ligase from HeLa cytoplasmic fraction (S100).
(A) In vitro ubiquitination of TBP by HeLa cell lysates. In vitro transcribed and translated HA-TBP was incubated with indicated HeLa cellular fractions for ubiquitination assays. After the …
Figure 2—figure supplement 1
GST-TBP can be used as a substrate for in vitro ubiquitination assays.
(A) GST-TBP but not GST is ubiquitinated by HeLa S100 cytoplasmic fraction in vitro. Recombinant GST-TBP and GST proteins (right) were incubated with Hela S100 fraction for in vitro ubiquitination …
Figure 3 with 2 supplements
In vitro ubiquitination of TBP using recombinant Huwe1 protein.
(A) Recombinant Huwe1 protein ubiquitinates TBP in vitro. Titrations (twofold concentration range) of recombinant His-tag Huwe1 protein are incubated with GST-TBP supplemented with E1, UbcH5b, …
Figure 3—figure supplement 1
Huwe1 mediates TBP ubiquitination at multiple sites.
(A) PageBlue SDS-PAGE Gel of purified recombinant wild-type (WT), catalytic domain truncation (ΔC) and catalytic site C4341S mutant (C → S) Huwe1 proteins are shown. HiMark pre-stained protein …
Figure 3—figure supplement 2
Huwe1 directly interacts with TBP in vitro.
GST-tagged full length (FL) TBP, TBP C-terminal DNA binding domain (DBD) and N-terminal transactivation domain (TAD) were used to pull-down purified recombinant Huwe1 protein. Pull down materials …
Figure 4 with 1 supplement
Huwe1 regulates TBP protein levels in proliferating cells.
(A) Huwe1 knockdown increases TBP protein levels in 293T cells. Western blot analysis of whole cell extracts from control (NT) and Huwe1 knockdown 293T cells was done using antibodies against Huwe1 …
Figure 4—figure supplement 1
Huwe1 knockdown prolongs half-life of fluorescent-timer TBP fusion protein in 273T cells as measured by fluoresent-timer assays.
(A) Schematic representation of the principle for fluorescent timer assay. (B) Fluorescent-timer TBP fusion protein exhibits higher mCherry/GFP ratio in Huwe1 knockdown 293T cells. 293T cells were …
Figure 5 with 1 supplement
Huwe1 regulates TBP protein level in myoblasts and myotubes.
(A) Lentivirus-mediated depletion of Huwe1 elevates TBP protein level in C2C12 myotubes. Cytoplasmic and nuclear extracts of C2C12 myoblasts infected with control (NT) lentiviruses or two different …
Figure 5—figure supplement 1
Huwe1 knockdown increases TBP protein half-life in C2C12 myoblasts.
(A) Control and Huwe1 knockdown C2C12 myoblasts were treated with 50 μg/ml cycloheximide. Cells were collected at indicated time points and analyzed by western blot using anti-TBP and anti-Tubulin …
Figure 6 with 2 supplements
Huwe1 is required for muscle differentiation and maintenance of normal muscle morphologies.
(A) Huwe1 knockdown impaired C2C12 differentiation efficiency. Shown are phase contrast images of control and Huwe1 knockdown C2C12 myoblasts after 6 days of differentiation in 2% horse serum. (B) …
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Figure 6—source data 1
Differentiation gene expression analysis of control and Huwe1 knockdown C2C12 cells on differentiation Day 6 by RNA-seq.
- https://doi.org/10.7554/eLife.08536.015
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Figure 6—source data 2
Differentiation gene expression analysis of control and Huwe1 knockdown myotubes on differentiation Day 8 by RNA-seq.
- https://doi.org/10.7554/eLife.08536.016
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Figure 6—source data 3
Differentiation gene expression analysis of control and Huwe1 knockdown C2C12 cells on differentiation Day 0 by RNA-seq.
- https://doi.org/10.7554/eLife.08536.017
Figure 6—figure supplement 1
Huwe1 knockdown causes modest changes in the transcriptional program of C2C12 myoblasts.
(A) Huwe1 knockdown C2C12 myoblasts did not exhibit significant morphological differences compared to control C2C12 s when cultured in proliferation medium (10% FBS). (B) Huwe1 knockdown results in …
Figure 6—figure supplement 2
Huwe1 knockdown inhibits the induction of master myogenic factors during C2C12 differentiation.
(A) Huwe1, Myod1 and Mef2c mRNA levels in control and two different Huwe1 knockdown C2C12 myoblast lines were analyzed by RT-qPCR and normalized to Gapdh. Shown are values relative to control cells …
Figure 7 with 2 supplements
Ubiquitin-specific protease 10 (USP10) regulates TBP protein level during myogenesis.
(A) USP10 overexpression inhibits TBP ubiquitination in vivo. 293T cells were transfected with indicated plasmids as well as a plasmid expressing HA-TBP. Ubiquitin conjugates were purified using …
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Figure 7—source data 1
- https://doi.org/10.7554/eLife.08536.021
Figure 7—figure supplement 1
USP10 overexpression elevates TBP protein levels and impairs differentiation capacity.
(A) USP10 overexpression increases TBP protein level. Wild type and USP10 overexpressing C2C12 (stable cell line) were double-stained using rabbit anti-USP10 and mouse anti-TBP primary antibodies, …
Figure 7—figure supplement 2
USP10 overexpression increases TBP protein half-life in C2C12 myoblasts.
(A) USP10 overexpressing C2C12 myoblasts were treated with 50 μg/ml cycloheximide. Cells were collected at indicated time points and analyzed by western blot using anti-TBP and anti-Tubulin …
Figure 8
Schematic representation of coordinated regulation of TBP protein level in proliferating and differentiated cells by E3 ligase Huwe1 and deubiquitinase USP10.
Both Huwe1 E3 ligase and USP10 deubiquitinase are present in proliferating myoblasts to maintain steady state TBP protein levels. Upon stimulation by differentiation signals, Huwe1 protein levels …
