Figure 1B . ITC binding assays of the UVR8 and HY5 VP - peptides versus the COP1 WD40 domain . The top panel represents the heats detected during each injection . The bottom panel represents the integrated heats of each injection , fitted to a one - site binding model ( solid line ) . The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1 ( 2500 μM in 175 μM ) ; HY5 - COP1 ( 1500 μM in 175 μM ) . The insets show the dissociation constants ( Kd ) and stoichiometries of binding ( N ) ( ± standard deviation ) . E . ITC assays of the HY5 and UVR8 VP - peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used ( titrant into cell ) : UVR8 - COP1Lys422Ala ( 1000 μM in 100 μM ) ; UVR8 - COP1Tyr441Ala ( 2500 μM in 90 μM ) ; UVR8 - COP1Trp467Ala ( 2500 μM in 90 μM ) ; HY5 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; HY5 - COP1Tyr441Ala ( 1600 μM in 138 μM ) ; HY5 - COP1Trp467Ala ( 1600 μM in 112 μM ) . The insets show the dissociation constants ( Kd ) . The stoichiometries of binding ( N ) for UVR8 - COP1Lys422Ala = 0 . 87 ± 0 . 2 ; HY5 - COP1Lys422Ala = 0 . 83 ± 0 . 2 ; HY5 - COP1Tyr441Ala = 0 . 92 ± 0 . 3 ( All measurements ± standard deviation ; n . d . : no detectable binding ) . Images of representative individuals ( F ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Data Information , lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines . quantification of hypocotyl lengths ( G ) of 4 - day - old seedlings grown with or without supplemental UV - B . The scale bar for all lines represent 5 mm except for cop1 - 5 where the scale bars represent 1 mm . Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines . Quantitative real - time PCR analysis of ( H ) HY5 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information : lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines . Quantitative real - time PCR analysis of ( I ) RUP2 expression . Four - day - old seedlings grown in white light were exposed to narrowband UV - B for 2 hours ( + UV - B ) , or not ( - UV - B ) . Error bars represent SEM of 3 biological replicates . Data Information lines used : wild - type ( Ws ) , uvr8 - 7 , cop1 - 4 , cop1 - 5 / Pro35S : YFP - COP1 ( WT ) , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala , cop1 - 5 / Pro35S : YFP - COP1Trp467Ala and cop1 - 5 . # 1 and # 2 : independent transgenic lines .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. ITC binding assays of the UVR8 and HY5 VP-peptides versus the COP1 WD40 domain. The top panel represents the heats detected during each injection. The bottom panel represents the integrated heats of each injection, fitted to a one-site binding model (solid line). The following concentrations were typically used (titrant into cell): UVR8 - COP1 (2500 μM in 175 μM); HY5 - COP1 (1500 μM in 175 μM). The insets show the dissociation constants (Kd) and stoichiometries of binding (N) (± standard deviation).E. ITC assays of the HY5 and UVR8 VP-peptides versus the different COP1 WD40 domain mutants The following concentrations were typically used (titrant into cell): UVR8 - COP1Lys422Ala (1000 μM in 100 μM); UVR8 - COP1Tyr441Ala (2500 μM in 90 μM); UVR8 - COP1Trp467Ala (2500 μM in 90 μM); HY5 - COP1Lys422Ala (1500 μM in 175 μM); HY5 - COP1Tyr441Ala (1600 μM in 138 μM); HY5 - COP1Trp467Ala (1600 μM in 112 μM). The insets show the dissociation constants (Kd). The stoichiometries of binding (N) for UVR8 - COP1Lys422Ala = 0.87 ± 0.2; HY5 - COP1Lys422Ala = 0.83 ± 0.2; HY5 - COP1Tyr441Ala = 0.92 ± 0.3 (All measurements ± standard deviation; n.d.: no detectable binding).Images of representative individuals (F) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Data Information , lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.quantification of hypocotyl lengths (G) of 4-day-old seedlings grown with or without supplemental UV-B. The scale bar for all lines represent 5 mm except for cop1-5 where the scale bars represent 1 mm. Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.Quantitative real-time PCR analysis of (H) HY5 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information: lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.Quantitative real-time PCR analysis of (I) RUP2 expression. Four-day-old seedlings grown in white light were exposed to narrowband UV-B for 2 hours (+UV-B), or not (-UV-B). Error bars represent SEM of 3 biological replicates. Data Information lines used: wild-type (Ws), uvr8-7, cop1-4, cop1-5/Pro35S:YFP-COP1 (WT), cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala, cop1-5/Pro35S:YFP-COP1Trp467Ala and cop1-5. #1 and #2: independent transgenic lines.


Figure 2A . ITC assay between the COP1 WD40 domain and full - length UVR8 pre - monomerized by UV - B light . Integrated heats are shown in solid , purple squares . For comparison , an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain ( from Fig 1B ) is also shown in open , purple squares . The following concentrations were typically used ( titrant into cell ) : COP1 - UVR8 + UV - B ( 130 μM in 20 μM ) . The inset shows the dissociation constant ( Kd ) , stoichiometry of binding ( N ) ( ± standard deviation ) . B . Binding kinetics of UVR8 pre - monomerized by UV - B versus the COP1 WD40 domain obtained by grating - coupled interferometry ( GCI ) . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8 + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant . D . Binding kinetics of UVR8ValPro / AlaAla pre - monomerized by UV - B versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8ValPro / AlaAla + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant . E . Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of HY5 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - HY5 ( highest concentration 2 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant . F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8 . ( Top ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Bottom ) representative filter lift assays . AD , activation domain ; BD , DNA binding domain ; Met , methionine .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. ITC assay between the COP1 WD40 domain and full-length UVR8 pre-monomerized by UV-B light. Integrated heats are shown in solid, purple squares. For comparison, an ITC experiment between UVR8 VP peptide and the COP1 WD40 domain (from Fig 1B) is also shown in open, purple squares. The following concentrations were typically used (titrant into cell): COP1 - UVR8 +UV-B (130 μM in 20 μM). The inset shows the dissociation constant (Kd), stoichiometry of binding (N) (± standard deviation).B. Binding kinetics of UVR8 pre-monomerized by UV-B versus the COP1 WD40 domain obtained by grating-coupled interferometry (GCI). Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.D. Binding kinetics of UVR8ValPro/AlaAla pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8ValPro/AlaAla +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.E. Binding kinetics of HY5 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of HY5 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - HY5 (highest concentration 2 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.F. Yeast 3-hybrid analysis of the COP1 - HY5 interaction in the presence of UVR8. (Top) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Bottom) representative filter lift assays. AD, activation domain; BD, DNA binding domain; Met, methionine.


Figure 3B . Representative image showing the phenotype of wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 seedlings grown for 4 days under white light ( - UV - B ) or white light supplemented with UV - B ( + UV - B ) . The scale bar represents 5 mm . C . Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings ; upper and lower hinges correspond to the first and third quartiles , the horizontal line in the interior of the box indicates the median . D . Anthocyanin accumulation in seedlings Average and SEM are shown ( n = 3 ) . E . Quantitative real - time PCR analysis of RUP2 , CHS , HY5 and ELIP2 expression in wild - type ( Ws ) , uvr8 - 7 and uvr8 - 7 / Pro35S : UVR8HY5C44 , uvr8 - 7 / Pro35S : UVR8HY5VP and uvr8 - 7 / Pro35S : UVR8TRIB1 4 - day old seedlings grown under white light in response to 2h of UV - B ( + UV - B vs . - UV - B ) . Error bars represent SEM of 3 biological replicates . Note : the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera . F . Yeast 3 - hybrid analysis of the COP1 - HY5 interaction in the presence of the UVR8HY5C44 chimera . ( Left pane ) Normalized Miller Units were calculated as a ratio of β - galactosidase activity in yeast grown under UV - B ( + UV - B ) versus yeast grown without UV - B ( - UV - B ) . Additionally , normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine , corresponding to induction ( - Met ) or repression ( + Met ) of Met25 promoter - driven UVR8 expression , respectively . Means and SEM for 3 biological repetitions are shown . ( Right panel ) Representative filter lift assays of the yeast analyzed in left panel . AD , activation domain ; BD , DNA binding domain ; Met , methionine . G . Binding kinetics of the UVR8HY5C44 chimera pre - monomerized by UV - B versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of UVR8HY5C44 injected are shown in red , with their respective 1 : 1 binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - UVR8HY5C44 + UV - B ( highest concentration 2 μM ) . ka = association rate constant , kd = disassociation rate constant , Kd = dissociation constant . H . Size - exclusion chromatography assay of purified , recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV - B .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Representative image showing the phenotype of wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 seedlings grown for 4 days under white light (- UV-B) or white light supplemented with UV-B (+ UV-B). The scale bar represents 5 mm.C. Quantification of hypocotyl length data Violin and box plots are shown for n > 60 seedlings; upper and lower hinges correspond to the first and third quartiles, the horizontal line in the interior of the box indicates the median.D. Anthocyanin accumulation in seedlings Average and SEM are shown (n = 3).E. Quantitative real-time PCR analysis of RUP2, CHS, HY5 and ELIP2 expression in wild-type (Ws), uvr8-7 and uvr8-7/Pro35S:UVR8HY5C44, uvr8-7/Pro35S:UVR8HY5VP and uvr8-7/Pro35S:UVR8TRIB1 4-day old seedlings grown under white light in response to 2h of UV-B (+UV-B vs. -UV-B). Error bars represent SEM of 3 biological replicates. Note: the primers used to detect the HY5 transcript abundance also bind to an identical region present in UVR8HY5C44 chimera.F. Yeast 3-hybrid analysis of the COP1-HY5 interaction in the presence of the UVR8HY5C44 chimera. (Left pane) Normalized Miller Units were calculated as a ratio of β-galactosidase activity in yeast grown under UV-B (+ UV-B) versus yeast grown without UV-B (- UV-B). Additionally, normalized Miller Units are reported separately here for yeast grown on media without or with 1 mM methionine, corresponding to induction (- Met) or repression (+ Met) of Met25 promoter-driven UVR8 expression, respectively. Means and SEM for 3 biological repetitions are shown. (Right panel) Representative filter lift assays of the yeast analyzed in left panel. AD, activation domain; BD, DNA binding domain; Met, methionine.G. Binding kinetics of the UVR8HY5C44 chimera pre-monomerized by UV-B versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of UVR8HY5C44 injected are shown in red, with their respective 1:1 binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - UVR8HY5C44 +UV-B (highest concentration 2 μM). ka = association rate constant, kd = disassociation rate constant, Kd = dissociation constant.H. Size-exclusion chromatography assay of purified, recombinant chimeric proteins expressed in Sf9 insect cells in the presence and absence of UV-B.


Figure 4A . Table summarizing affinities ( Kd , dissociation constant ) of VP - containing peptides versus the COP1 WD40 domain ( WT ) and COP1Lys422Ala as determined by ITC . All values are μM ( ± standard deviation ) . D . ITC assays between the COL3 VP - peptide ( left ) and CRY1 VP - peptide ( right ) versus the COP1 WD40 and COP1Lys422Ala , with a table summarizing their corresponding affinities . The following concentrations were typically used ( titrant into cell ) : COL3 - COP1 ( 2240 μM in 195 μM ) ; COL3 - COP1Lys422Ala ( 1500 μM in 175 μM ) ; CRY1 - COP1 ( 750 μM in 75 μM ) ; CRY1 - COP1Lys422Ala ( 1500 μM in 175 μM ) . The inset shows the dissociation constant ( Kd ) . The stoichiometries of binding ( N ) for COL3 - COP1 = 0 . 75 ± 0 . 2 ; COL3 - COP1Lys422Ala = 0 . 90 ± 0 . 2 ; CRY1 - COP1 = 0 . 99 ± 0 . 3 ; CRY1 - COP1Lys422Ala = 0 . 98 ± 0 . 1 ( All measurements ± standard deviation ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Table summarizing affinities (Kd, dissociation constant) of VP-containing peptides versus the COP1 WD40 domain (WT) and COP1Lys422Ala as determined by ITC. All values are μM (± standard deviation).D. ITC assays between the COL3 VP-peptide (left) and CRY1 VP-peptide (right) versus the COP1 WD40 and COP1Lys422Ala, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): COL3 - COP1 (2240 μM in 195 μM); COL3 - COP1Lys422Ala (1500 μM in 175 μM); CRY1 - COP1 (750 μM in 75 μM); CRY1 - COP1Lys422Ala (1500 μM in 175 μM). The inset shows the dissociation constant (Kd). The stoichiometries of binding (N) for COL3 - COP1 = 0.75 ± 0.2; COL3 - COP1Lys422Ala = 0.90 ± 0.2; CRY1 - COP1 = 0.99 ± 0.3; CRY1 - COP1Lys422Ala = 0.98 ± 0.1 (All measurements ± standard deviation).


Figure 5A . Representative image of wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 , cop1 - 5 / Pro35S : YFP - COP1Lys422Ala , cop1 - 5 / Pro35S : YFP - COP1Tyr441Ala and cop1 - 5 / Pro35S : YFP - COP1Trp467Ala transgenic lines grown for 39 days in long day conditions . B . Representative image of individual wild - type ( Ws ) , co - 11 , cop1 - 5 / Pro35S : YFP - COP1 and cop1 - 5 / Pro35S : YFP - COP1Lys422Ala plants grown for 39 days in long days conditions . C . Quantification of flowering time . Means and SD are shown ( n = 14 ) . D . Number of rosette and cauline leaves at flowering . Means and SD are shown ( n = 14 ) . E . Immunoblot analysis of CRY2 and actin ( loading control ) protein levels in wild - type ( Col ) , cry2 - 1 , wild - type ( Ws ) , cop1 - 5 / Pro35S : YFP - COP1 and cop1 - 5 / Pro35S : YFP - COP1Lys422Ala seedlings grown for 4 days in darkness . F . ITC assays between the CO VP - peptide ( left ) and CRY2 VP - peptide ( right ) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains , with a table summarizing their corresponding affinities . The following concentrations were typically used ( titrant into cell ) : CO - COP1 ( 2240 μM in 195 μM ) ; CO - COP1Lys422Ala ( 1200 μM in 120 μM ) ; CRY2 - COP1 ( 750 μM in 70 μM ) ; CRY2 - COP1Lys422Ala ( 3000 μM in 175 μM ) . The stoichiometries of binding ( N ) for CO - COP1 = 1 . 03 ± 0 . 00 ; CO - COP1Lys422Ala = 0 . 89 ± 0 . 03 ; CRY2 - COP1 = 1 . 04 ± 0 . 20 ; CRY2 - COP1Lys422Ala = 1 ( fixed ) ( All measurements ± standard deviation ) . G , H . Binding kinetics of the ( G ) full - length activated CRY2 and ( H ) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments . Sensorgrams of protein injected are shown in red , with their respective heterogenous ligand binding model fits in black . The following amounts were typically used : ligand - COP1 ( 2000 pg / mm2 ) ; analyte - CRY2 ( highest concentration 14 μM ) , CRY2NC80 ( highest concentration 450 μM ) . ka = association rate constant , kd = dissociation rate constant , Kd = dissociation constant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative image of wild-type (Ws), cop1-5/Pro35S:YFP-COP1, cop1-5/Pro35S:YFP-COP1Lys422Ala, cop1-5/Pro35S:YFP-COP1Tyr441Ala and cop1-5/Pro35S:YFP-COP1Trp467Ala transgenic lines grown for 39 days in long day conditions.B. Representative image of individual wild-type (Ws), co-11, cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala plants grown for 39 days in long days conditions.C. Quantification of flowering time. Means and SD are shown (n = 14). D. Number of rosette and cauline leaves at flowering. Means and SD are shown (n = 14). E. Immunoblot analysis of CRY2 and actin (loading control) protein levels in wild-type (Col), cry2-1, wild-type (Ws), cop1-5/Pro35S:YFP-COP1 and cop1-5/Pro35S:YFP-COP1Lys422Ala seedlings grown for 4 days in darkness.F. ITC assays between the CO VP-peptide (left) and CRY2 VP-peptide (right) versus the COP1 WD40 and the COP1Lys422Ala WD40 domains, with a table summarizing their corresponding affinities. The following concentrations were typically used (titrant into cell): CO - COP1 (2240 μM in 195 μM); CO - COP1Lys422Ala (1200 μM in 120 μM); CRY2 - COP1 (750 μM in 70 μM); CRY2 - COP1Lys422Ala (3000 μM in 175 μM). The stoichiometries of binding (N) for CO - COP1 = 1.03 ± 0.00; CO - COP1Lys422Ala = 0.89 ± 0.03; CRY2 - COP1 = 1.04 ± 0.20; CRY2 - COP1Lys422Ala = 1 (fixed) (All measurements ± standard deviation).G, H. Binding kinetics of the (G) full-length activated CRY2 and (H) CRY2NC80 versus the COP1 WD40 domain obtained by GCI experiments. Sensorgrams of protein injected are shown in red, with their respective heterogenous ligand binding model fits in black. The following amounts were typically used: ligand - COP1 (2000 pg/mm2); analyte - CRY2 (highest concentration 14 μM), CRY2NC80 (highest concentration 450 μM). ka = association rate constant, kd = dissociation rate constant, Kd = dissociation constant.


Figure 1A ) Auto - Ub of recombinant CHN - 1 and CHN - 1 - UFD - 2 was carried out using the E2 UBE2D1 . Time - dependent ( 0 , 10 , 20 , 40 , 80 , 120 min ) CHN - 1 auto - Ub was performed as indicated using wild - type ubiquitin . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Below , a graph representing the nmol of ubiquitylated CHN - 1 vs . time for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2 ( cyan ) . Plotted data are the mean of three technical replicates . Error bars represent the standard error of measurement ( SEM ) ; statistical and significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ CHN - 1 vs Time , CHN - 1 + UFD - 2 vs Time , and CHN - 1 vs CHN - 1 + UFD - 2 with increasing time ] . B ) CHN - 1 auto - Ub was performed in the presence of UFD - 2 with the increasing molar concentration as indicated . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , signal quantification of the unmodified CHN - 1 ( yellow ) , ubiquitylated fraction < 70 kDa ( cyan ) and > 70 kDa ( magenta ) , plotted as a percentage of different CHN - 1 species present in the indicated condition . Plotted data are the mean from the three technical replicates . Error bars represent the SEM ; statistical significance was determined using a two - way ANOVA test . ( * * P < 0 . 01 ) C ) Auto - Ub was performed as indicated using recombinant CHN - 1 and UFD - 2P951A , UBE2D1 E2 , UbWT , UbNoK , or Ub with substitutions of lysines 29 , 48 , and 63 to arginines ( Ub3KTR ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . E ) CHN - 1 auto - Ub was performed in the presence of recombinant C . elegans UFD - 2 and S . cerevisiae Ufd - 2p and UBE2D1 . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Auto-Ub of recombinant CHN-1 and CHN-1-UFD-2 was carried out using the E2 UBE2D1. Time-dependent (0, 10, 20, 40, 80, 120 min) CHN-1 auto-Ub was performed as indicated using wild-type ubiquitin. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Below, a graph representing the nmol of ubiquitylated CHN-1 vs. time for CHN-1 alone (black) or CHN-1+UFD-2 (cyan). Plotted data are the mean of three technical replicates. Error bars represent the standard error of measurement (SEM); statistical and significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [CHN-1 vs Time, CHN-1+UFD-2 vs Time, and CHN-1 vs CHN-1+UFD-2 with increasing time].B) CHN-1 auto-Ub was performed in the presence of UFD-2 with the increasing molar concentration as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, signal quantification of the unmodified CHN-1 (yellow), ubiquitylated fraction <70 kDa (cyan) and >70 kDa (magenta), plotted as a percentage of different CHN-1 species present in the indicated condition. Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using a two-way ANOVA test. (**P < 0.01)C) Auto-Ub was performed as indicated using recombinant CHN-1 and UFD-2P951A, UBE2D1 E2, UbWT, UbNoK, or Ub with substitutions of lysines 29, 48, and 63 to arginines (Ub3KTR). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.E) CHN-1 auto-Ub was performed in the presence of recombinant C. elegans UFD-2 and S. cerevisiae Ufd-2p and UBE2D1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


Figure 2B ) CHN - 1 auto - Ub was performed as indicated using increasing molar concentrations ( 0 . 5 , 1 , 2 , 3 , 4 µM ) of UBE2D1 without and with a complexing equimolar concentration ( 1 : 1 ) of recombinant CHN - 1 and UFD - 2P951A ( 1 . 5 µM CHN - 1 with 1 . 5 µM of recombinant UFD - 2P951A ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , a graph representing the nmol of ubiquitylated CHN - 1 vs . UBE2D1 ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) . Plotted data are the mean from the three technical replicates . Error bars represent the SEM ; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ nmol of ubiquitylated CHN - 1 vs UBE2D1 ( µM ) in the presence of CHN - 1 or CHN - 1 + UFD - 2P951A and CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 ( µM ) ] . C ) Discharging assay of Ub - charged UBE2D1 was carried out in the presence of CHN - 1 or CHN - 1 - UFD - 2P951A . The experimental sample was run together with the control with and without a reducing agent ( 50 mM DTT ) . The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer . Proteins were resolved via SDS - PAGE and immunoblotted with anti - UBE2D1 antibodies . Below , quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) . The graph representing the UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) and Plotted data are the mean of three technical replicates . Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ UBE2D1 vs UBE2D1 - Ub ( µM ) in the presene of CHN - 1 or CHN - 1 + UFD - 2P951A and CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 - Ub ( µM ) ] . D ) CHN - 1 auto - Ub was performed as indicated in the presence of Ube2W - Ub or Ube2W - UbFLAG without and with equimolar concentration of recombinant CHN - 1 and UFD - 2P951A . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . For each sample , the quantified relative signal after probing the blot using anti - CHN - 1 antibodies is shown as a ratio above the respective signal . Below , a schematic of the CHN - 1 - UbFLAG ( cyan ) , CHN - 1 - Ub ( magenta ) , and unmodified CHN - 1 signal ( black ) presented as the ratio and the signal fold change between CHN - 1 and CHN - 1 - UFD - 2P951A .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B) CHN-1 auto-Ub was performed as indicated using increasing molar concentrations (0.5, 1, 2, 3, 4 µM) of UBE2D1 without and with a complexing equimolar concentration (1:1) of recombinant CHN-1 and UFD-2P951A (1.5 µM CHN-1 with 1.5 µM of recombinant UFD-2P951A). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, a graph representing the nmol of ubiquitylated CHN-1 vs. UBE2D1 (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan). Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [nmol of ubiquitylated CHN-1 vs UBE2D1(µM) in the presence of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1(µM)].C) Discharging assay of Ub-charged UBE2D1 was carried out in the presence of CHN-1 or CHN-1-UFD-2P951A. The experimental sample was run together with the control with and without a reducing agent (50 mM DTT). The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer. Proteins were resolved via SDS-PAGE and immunoblotted with anti-UBE2D1 antibodies. Below, quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs. UBE2D1-Ub (µM). The graph representing the UBE2D1 fraction vs. UBE2D1-Ub (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan) and Plotted data are the mean of three technical replicates. Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [UBE2D1 vs UBE2D1-Ub(µM) in the presene of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1-Ub(µM)].D) CHN-1 auto-Ub was performed as indicated in the presence of Ube2W-Ub or Ube2W-UbFLAG without and with equimolar concentration of recombinant CHN-1 and UFD-2P951A. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. For each sample, the quantified relative signal after probing the blot using anti-CHN-1 antibodies is shown as a ratio above the respective signal. Below, a schematic of the CHN-1-UbFLAG (cyan), CHN-1-Ub (magenta), and unmodified CHN-1 signal (black) presented as the ratio and the signal fold change between CHN-1 and CHN-1-UFD-2P951A.


Figure 3A ) Auto - Ub of recombinant CHN - 1 using UBE2D1 was performed alone or complexed with UFD - 2 in the presence of recombinant DAF - 21 , DAF - 21ΔEEVD , HSP - 1 , or HSP - 1ΔEEVD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . B ) Top , schematics of the HSP - 1 peptide sequence ( cyan ) aligned with the C - terminal sequence of full - length HSP - 1 ( amino acids 600 - 640 ) used in the ubiquitylation reaction . Auto - Ub of recombinant CHN - 1 was performed as indicated HSP - 1 ‑ derived peptide . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) or CHN - 1 + HSP - 1 peptide ( cyan ) . Plotted data are the mean of three technical replicates . C ) Top , schematics of the UFD - 2 peptide sequences aligned with the C - terminal sequence of full - length UFD - 2 ( UFD - 2 amino acids 864 - 914 ) for PEPTIDE 1 ( yellow ) and PEPTIDE 2 ( cyan ) and ( UFD - 2 amino acids 648 - 678 ) for PEPTIDE 3 ( magenta ) used in the ubiquitylation reaction . Below , auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of UFD - 2 ‑ derived peptides . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 + PEPTIDE 1 ( yellow ) , CHN - 1 + PEPTIDE 2 ( cyan ) , or CHN - 1 + PEPTIDE 3 ( magenta ) . Plotted data are the mean of three technical replicates . D ) Auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of recombinant HSP - 1 or HSP - 1EEYD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . E ) Auto - Ub of recombinant CHN - 1 was performed as indicated alone or with UFD - 2 or UFD - 2EEVD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . F ) CHN - 1R230A auto - Ub was performed as indicated in the presence of recombinant UFD - 2 and HSP - 1 . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Auto-Ub of recombinant CHN-1 using UBE2D1 was performed alone or complexed with UFD-2 in the presence of recombinant DAF-21, DAF-21ΔEEVD, HSP-1, or HSP-1ΔEEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.B) Top, schematics of the HSP-1 peptide sequence (cyan) aligned with the C-terminal sequence of full-length HSP-1 (amino acids 600-640) used in the ubiquitylation reaction. Auto-Ub of recombinant CHN-1 was performed as indicated HSP-1‑derived peptide. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black) or CHN-1+HSP-1 peptide (cyan). Plotted data are the mean of three technical replicates.C) Top, schematics of the UFD-2 peptide sequences aligned with the C-terminal sequence of full-length UFD-2 (UFD-2 amino acids 864-914) for PEPTIDE 1 (yellow) and PEPTIDE 2 (cyan) and (UFD-2 amino acids 648-678) for PEPTIDE 3 (magenta) used in the ubiquitylation reaction. Below, auto-Ub of recombinant CHN-1 was performed as indicated in the presence of UFD-2‑derived peptides. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1+PEPTIDE 1 (yellow), CHN-1+PEPTIDE 2 (cyan), or CHN-1+PEPTIDE 3 (magenta). Plotted data are the mean of three technical replicates.D) Auto-Ub of recombinant CHN-1 was performed as indicated in the presence of recombinant HSP-1 or HSP-1EEYD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.E) Auto-Ub of recombinant CHN-1 was performed as indicated alone or with UFD-2 or UFD-2EEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.F) CHN-1R230A auto-Ub was performed as indicated in the presence of recombinant UFD-2 and HSP-1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


Figure 4A ) To detect CHN - 1 and UFD - 2 , indicated lysates of young adult worms were subjected to immunoblotting with anti - CHN - 1 and anti - UFD - 2 antibodies . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels and plotted as N2 ( wild - type ; black ) , ufd - 2 ( tm1380 ) ( cyan ) , and UFD - 2P951A ( magenta ) . Plotted data are the mean of three biological replicates . B ) CHN - 1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor ( MG - 132 , 10µM ) and DUB inhibitor ( NEM , 100mM ) . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels plotted as control ( black ) , or MG - 132 treated ( magenta ) in N2 ( wild - type ) and ufd - 2 ( tm1380 ) . Plotted data are the mean of three biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) To detect CHN-1 and UFD-2, indicated lysates of young adult worms were subjected to immunoblotting with anti-CHN-1 and anti-UFD-2 antibodies. Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels and plotted as N2 (wild-type; black), ufd-2(tm1380) (cyan), and UFD-2P951A (magenta). Plotted data are the mean of three biological replicates.B) CHN-1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor (MG-132, 10µM) and DUB inhibitor (NEM, 100mM). Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels plotted as control (black), or MG-132 treated (magenta) in N2 (wild-type) and ufd-2(tm1380). Plotted data are the mean of three biological replicates.


Figure 5B ) Ubiquitylation of recombinant AHCY - 1 was performed as indicated . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Right , quantification of the AHCY - 1 modifications ( unmodified , oligo - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 - UFD - 2 ( magenta ) , or CHN - 1 - UFD - 2EEVD ( cyan ) . Plotted data are the mean of three technical replicates . C ) Protein level of endogenous AHCY - 1 in N2 ( wild - type ) , chn - 1 ( by155 ) , CHN - 1 OE , and ufd - 2 ( tm1380 ) young adult worms treated with the proteasome inhibitor ( MG - 132 , 10 µM ) and DUB inhibitor ( NEM , 100 mM ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Tubulin served as a loading control . Right , quantification of the modified AHCY - 1 signals plotted as Ub - modified AHCY - 1 species normalized to unmodified endogenous AHCY - 1 signal and plotted for N2 ( wild - type ; black ) , chn - 1 ( by155 ) ( magenta ) , CHN - 1 OE ( yellow ) , and ufd - 2 ( tm1380 ) ( cyan ) . Plotted data are the mean of three biological replicates . D ) Representative images of GFP : : AHCY - 1 fluorescence in chn - 1 ( by155 ) , ufd - 2 ( tm1380 ) , and CHN - 1 OE background . Scale bar = 200µm . Below , quantification of the AHCY - 1 GFP signal plotted as fluorescence intensity for GFP : : AHCY - 1 expressing worms ( control ; black ) , chn - 1 ( by155 ) ( magenta ) , ufd - 2 ( tm1380 ) ( cyan ) or CHN - 1 OE ( yellow ) . Plotted data are the mean of three biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B) Ubiquitylation of recombinant AHCY-1 was performed as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Right, quantification of the AHCY-1 modifications (unmodified, oligo-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1-UFD-2 (magenta), or CHN-1-UFD-2EEVD (cyan). Plotted data are the mean of three technical replicates.C) Protein level of endogenous AHCY-1 in N2 (wild-type), chn-1(by155), CHN-1 OE, and ufd-2(tm1380) young adult worms treated with the proteasome inhibitor (MG-132, 10 µM) and DUB inhibitor (NEM, 100 mM). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Tubulin served as a loading control. Right, quantification of the modified AHCY-1 signals plotted as Ub-modified AHCY-1 species normalized to unmodified endogenous AHCY-1 signal and plotted for N2 (wild-type; black), chn-1(by155) (magenta), CHN-1 OE (yellow), and ufd-2(tm1380) (cyan). Plotted data are the mean of three biological replicates.D) Representative images of GFP::AHCY-1 fluorescence in chn-1(by155), ufd-2(tm1380), and CHN-1 OE background. Scale bar = 200µm. Below, quantification of the AHCY-1 GFP signal plotted as fluorescence intensity for GFP::AHCY-1 expressing worms (control; black), chn-1(by155) (magenta), ufd-2(tm1380) (cyan) or CHN-1 OE (yellow). Plotted data are the mean of three biological replicates.


Figure 1Anti - Flag Western Blot for transiently transfected FlagSLC25A51 variants and empty vector control in HeLa cells ; HSP60 , loading control . In vitro pull down of FlagSLC25A51 variants using cardiolipin - bound or control beads and detected with anti - Flag Western Blots .

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Figure 1Anti-Flag Western Blot for transiently transfected FlagSLC25A51 variants and empty vector control in HeLa cells; HSP60, loading control.In vitro pull down of FlagSLC25A51 variants using cardiolipin-bound or control beads and detected with anti-Flag Western Blots.


Figure 2Free mitochondrial NAD + levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control ( n = 25 biological replicates ) , wildtype FlagSLC25A51 ( n = 25 biological replicates ) , and indicated mutants ( n = 4 - 6 biological replicates ) . Measurements were taken at 24 hours post - transfection ; the dashed line indicates the baseline defined by the empty vector control , and red denotes data equivalent to wildtype . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , ANOVA p < 0 . 0001 with post - hoc Dunnett ' s test compared to empty vector control , * p < 0 . 05 , * * p < 0 . 01 . ( bottom ) Protein expression from HeLa cells transiently transfected with empty vector control , wildtype FlagSLC25A51 and the indicated variants using anti - Flag western blot ; HSP60 , loading control . Quantitation of uptake of 32P - NAD + after 1 hour by E . coli cells expressing wildtype SLC25A51 ( amino acids 29 - 297 ) and the indicated mutants ; wildtype activity and equivalent , red . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , n = 5 - 8 biological replicates , ANOVA p < 0 . 0001 with post - hoc Dunnett ' s test compared to wildtype , * * * p < 0 . 001 .

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Figure 2Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 25 biological replicates), wildtype FlagSLC25A51 (n = 25 biological replicates), and indicated mutants (n = 4 - 6 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p <0.0001 with post-hoc Dunnett's test compared to empty vector control, *p < 0.05, **p < 0.01. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.Quantitation of uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and the indicated mutants; wildtype activity and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype, ***p < 0.001.


Figure 3Free mitochondrial NAD + levels measured using a ratiometric biosensor in HeLa cells co - expressing empty vector control ( n = 8 biological replicates ) , wildtype FlagSLC25A51 ( n = 8 biological replicates ) , and indicated mutants ( n = 4 biological replicates ) . Measurements were taken at 24 hours post - transfection ; the dashed line indicates the baseline defined by the empty vector control , and red denotes data equivalent to wildtype . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , ANOVA p < 0 . 0001 with post - hoc Dunnett ' s test compared to empty vector control * * * p < 0 . 001 . ( bottom ) Protein expression from HeLa cells transiently transfected with empty vector control , wildtype FlagSLC25A51 and the indicated variants using anti - Flag western blot ; HSP60 , loading control . Relative uptake of 32P - NAD + after 1 hour by E . coli cells expressing wildtype SLC25A51 ( amino acids 29 - 297 ) or the E132A mutant when competed by 100 μM ( n = 3 biological replicates ) and 250 μM ( n = 4 biological replicates ) unlabeled NAD + ( red ) or NADH ( black ) . Data is shown as floating bars with central line at mean and the box represents the min and max value , unpaired two - sided t - test * p < 0 . 05 . Quantitation of 32P - NAD + uptake after 1 hour in E . coli cells expressing wildtype SLC25A51 ( amino acids 29 - 297 ) and indicated mutants ; wildtype activity ( n = 19 biological replicates ) and equivalent , red . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , n = 5 - 8 biological replicates , ANOVA p < 0 . 0001 with post - hoc Dunnett ' s test compared to wildtype * * * p < 0 . 001 . Free mitochondrial NAD + levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control ( n = 27 biological replicates ) , wildtype FlagSLC25A51 ( n = 27 biological replicates ) , and indicated mutants ( n = 4 - 7 biological replicates ) . Measurements were taken at 24 hours post - transfection ; the dashed line indicates the baseline defined by the empty vector control , and red denotes data equivalent to wildtype . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , ANOVA p < 0 . 0001 , post - hoc Dunnett ' s test compared to empty vector control * * * p < 0 . 001 . ( bottom ) Protein expression from HeLa cells transiently transfected with empty vector control , wildtype FlagSLC25A51 and the indicated variants detected by anti - Flag western blot ; HSP60 , loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells co-expressing empty vector control (n = 8 biological replicates), wildtype FlagSLC25A51 (n = 8 biological replicates), and indicated mutants (n = 4 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001 with post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants using anti-Flag western blot; HSP60, loading control.Relative uptake of 32P-NAD+ after 1 hour by E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) or the E132A mutant when competed by 100 μM (n = 3 biological replicates) and 250 μM (n = 4 biological replicates) unlabeled NAD+ (red) or NADH (black). Data is shown as floating bars with central line at mean and the box represents the min and max value, unpaired two-sided t-test *p < 0.05.Quantitation of 32P-NAD+ uptake after 1 hour in E. coli cells expressing wildtype SLC25A51 (amino acids 29-297) and indicated mutants; wildtype activity (n = 19 biological replicates) and equivalent, red. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 5 - 8 biological replicates, ANOVA p <0.0001 with post-hoc Dunnett's test compared to wildtype ***p < 0.001.Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 27 biological replicates), wildtype FlagSLC25A51 (n = 27 biological replicates), and indicated mutants (n = 4 - 7 biological replicates). Measurements were taken at 24 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control ***p < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected by anti-Flag western blot; HSP60, loading control.


Figure 4Free mitochondrial NAD + levels measured using a ratiometric biosensor in HEK293 SLC25A51 KO cells expressing empty vector control , wildtype FlagSLC25A51 , and indicated mutants . Measurements were taken at 48 hours post - transfection ; the dashed line indicates the baseline defined by the empty vector control , and red denotes data equivalent to wildtype . Data are shown in box and whisker format , with hinges at 25th and 75th percentiles , whiskers represent min and max and the line is the median , n = 4 - 6 biological replicates , ANOVA p < 0 . 0001 , post - hoc Dunnett ' s test compared to empty vector control * * p < 0 . 01 , * * * p < 0 . 001 . ( bottom ) Protein expression from HEK293 SLC25A51 KO cells transiently transfected with empty vector control , wildtype FlagSLC25A51 and the indicated variants detected with anti - Flag western blot ; HSP60 , loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Free mitochondrial NAD+ levels measured using a ratiometric biosensor in HEK293 SLC25A51 KO cells expressing empty vector control, wildtype FlagSLC25A51, and indicated mutants. Measurements were taken at 48 hours post-transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25th and 75th percentiles, whiskers represent min and max and the line is the median, n = 4 - 6 biological replicates, ANOVA p < 0.0001, post-hoc Dunnett's test compared to empty vector control **p < 0.01, ***p < 0.001. (bottom) Protein expression from HEK293 SLC25A51 KO cells transiently transfected with empty vector control, wildtype FlagSLC25A51 and the indicated variants detected with anti-Flag western blot; HSP60, loading control.


Figure 1 ( C ) qRT - PCR expression of endogenous repeats following shRNA - mediated KAP1 depletion in NTERA - 2 cells . Results were normalized to β2 microglobulin ( B2M ) . KAP1 expression levels were verified by qRT - PCR and Western blot . A representative experiment of 2 experiments is shown . Two - tailed unpaired t tests were done ( HERVK14C _ 2 p - value = 0 . 002300 ) . ( D qRT - PCR expression of endogenous repeats following KAP1 knockout in HeLa Results were normalized to B2M . KAP1 expression levels were verified by qRT - PCR and Western blot . Two - tailed unpaired t tests were done and p - values are : HERVK14C _ 1 : 0 . 0017 , HERVK14C _ 2 : 0 . 0044 , SVA D VNTR : 0 . 0243 Clones 8 , 12 and 15 ( from D ) were selected for mRNA - sequencing . E ) qRT - PCR expression of endogenous repeats following KAP1 knockout in 293T cells Results were normalized to B2M . KAP1 expression levels were verified by qRT - PCR and Western blot . For ( E ) , we found that clone I ( also treated with KAP1 sgRNA ) retained KAP1 protein expression ( E , right panel ) so we only used clones II , III and IV to explore phenotype ( E , left panel ) , which represented validated KAP1 knockouts . Two - tailed unpaired t tests were done and p - values are : ( E ) HERVK14C _ 2 : < 0 . 0001 . ( F ) qRT - PCR expression of endogenous repeats following shRNA - mediated KAP1 depletion in PBMCs ( day 6 post - transduction ) . Results were normalized to B2M . All error bars show standard deviation ( SD ) . All numbers above bars depict fold changes compared to control cells ( to one decimal place ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in NTERA-2 cells. Results were normalized to β2 microglobulin (B2M). KAP1 expression levels were verified by qRT-PCR and Western blot. A representative experiment of 2 experiments is shown. Two-tailed unpaired t tests were done (HERVK14C_2 p-value = 0.002300).(D qRT-PCR expression of endogenous repeats following KAP1 knockout in HeLa Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. Two-tailed unpaired t tests were done and p-values are: HERVK14C_1: 0.0017, HERVK14C_2: 0.0044, SVA D VNTR: 0.0243 Clones 8, 12 and 15 (from D) were selected for mRNA-sequencing.E) qRT-PCR expression of endogenous repeats following KAP1 knockout in 293T cells Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. For (E), we found that clone I (also treated with KAP1 sgRNA) retained KAP1 protein expression (E, right panel) so we only used clones II, III and IV to explore phenotype (E, left panel), which represented validated KAP1 knockouts. Two-tailed unpaired t tests were done and p-values are: (E) HERVK14C_2: <0.0001.(F) qRT-PCR expression of endogenous repeats following shRNA-mediated KAP1 depletion in PBMCs (day 6 post-transduction). Results were normalized to B2M. All error bars show standard deviation (SD). All numbers above bars depict fold changes compared to control cells (to one decimal place).


Figure 2 ( A ) Boxplots showing repeats significantly upregulated ( > 2 fold where p = < 0 . 05 using DESeq2 ) in knockout ( N = 3 ) compared to wildtype ( N = 3 ) HeLa cells based on mRNA - sequencing data . P values = 0 . 0174 ( HERV - S ) , 0 . 0047 ( HERV - K14C ) , 0 . 00013 ( HERV - T LTR6B ) and 1 . 90E - 06 ( HERV - T HERVS71 ) . HERV - T and HERV - S but not HERVK14C also reached significance when only adjusted p - values were considered , where differences are compared to the whole of repBase . KAP1 binding ( ENCODE data ) is shown in Fig S1A . All numbers above bars depict fold changes compared to control cells ( to one decimal place ) . ( F ) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP - seq correlation analyses using ChIP - Cor software ( see methods ) . Each plot shows duplicate ChIP - seq experiments from ENCODE . See Fig EV3B for complete data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Boxplots showing repeats significantly upregulated (>2 fold where p=<0.05 using DESeq2) in knockout (N=3) compared to wildtype (N=3) HeLa cells based on mRNA-sequencing data. P values = 0.0174 (HERV-S), 0.0047 (HERV-K14C), 0.00013 (HERV-T LTR6B) and 1.90E-06 (HERV-T HERVS71). HERV-T and HERV-S but not HERVK14C also reached significance when only adjusted p-values were considered, where differences are compared to the whole of repBase. KAP1 binding (ENCODE data) is shown in Fig S1A. All numbers above bars depict fold changes compared to control cells (to one decimal place).(F) Genomic coordinates of the common KAP1 sites identified in Fig EV3D were subjected to ChIP-seq correlation analyses using ChIP-Cor software (see methods). Each plot shows duplicate ChIP-seq experiments from ENCODE. See Fig EV3B for complete data.


Figure 3 ( B ) NTERA - 2 cells were transduced with either an empty vector ( WT ) or the same vector containing an shRNA against KAP1 ( KD ) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors . GFP expression was analysed 72 hours post reporter transduction . A representative experiment of 2 experiments is shown . Two - tailed unpaired t test p - values : PBS - LTR - GFP : 0 . 0148 ; PBSChr15 - LTR - GFP : 0 . 0377 ( C ) The same as ( B ) except that here KAP1 wildtype ( WT ) and knockout ( KO ) 293T cells were used . P - values : PBS - LTR - GFP : 0 . 0044 ; PBSChr15 - LTR - GFP : 0 . 0077 . ( D ) PBMCs were transduced with increasing doses of GFP reporter vectors ( vectors were normalized to the same number of infectious units after titering them on permissive cells ) , left plot . Vectors were integrated at similar levels as measured by GFP Taqman qPCR , right plot . A 293T cell line with a single vector copy integrant ( PGK - GFP ) was used as a control to estimate the absolute copy numbers . A representative experiment of 3 experiments is shown here . Two - tailed unpaired t test p - value 0 . 0288 ( dose 3 ) . Error bars show SD .

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Figure 3(B) NTERA-2 cells were transduced with either an empty vector (WT) or the same vector containing an shRNA against KAP1 (KD) prior to puromycin selection and transduction with increasing doses of GFP reporter vectors. GFP expression was analysed 72 hours post reporter transduction. A representative experiment of 2 experiments is shown. Two-tailed unpaired t test p-values: PBS-LTR-GFP: 0.0148; PBSChr15-LTR-GFP: 0.0377(C) The same as (B) except that here KAP1 wildtype (WT) and knockout (KO) 293T cells were used. P-values: PBS-LTR-GFP: 0.0044; PBSChr15-LTR-GFP: 0.0077.(D) PBMCs were transduced with increasing doses of GFP reporter vectors (vectors were normalized to the same number of infectious units after titering them on permissive cells), left plot. Vectors were integrated at similar levels as measured by GFP Taqman qPCR, right plot. A 293T cell line with a single vector copy integrant (PGK-GFP) was used as a control to estimate the absolute copy numbers. A representative experiment of 3 experiments is shown here. Two-tailed unpaired t test p-value 0.0288 (dose 3). Error bars show SD.


Figure 4 ( A ) qRT - PCR expression of endogenous repeats ( left ) and ISGs ( right ) following shRNA - mediated KAP1 depletion in HeLa cells ( day 6 post transduction ) , normalized to B2M and GAPDH respectively . n = 2 and two - tailed unpaired t test p - values are : HERVK14C : 0 . 025 , 0 . 003 ; SVA VNTR : 0 . 00027 , 0 . 01 ; ISG56 : 0 . 002 ; CCL5 : 0 . 007 , 0 . 00003 ; CXCL10 : 0 . 0008 ; Mxa : 0 . 011 ; IKba : 0 . 008 , 0 . 01 . See also Fig S2AB . ( B ) qRT - PCR expression of endogenous repeats ( left ) and ISGs ( right ) following 5 - AZA treatment of HeLa cells and PBMCs ( day 6 post transduction ) . A representative experiment of two is shown in each case . Two - tailed unpaired t tests were performed . HeLa cell p - values : ISG56 : 0 . 005 ; CCL5 : 0 . 0006 ; CXCL10 : 0 . 0007 ; Mxa : 0 . 0001 ; IFNb : 0 . 02 . For repeat expression ( left panel ) , p values not available as N = 2 . PBMC p - values : HERVK14C : 0 . 1 ; SVA D VNTR : 0 . 003 ; ISG56 : 0 . 001 ; CCL5 : 0 . 012 ; CXCL10 : 0 . 012 ; Mxa : 0 . 02 ; IKba : 0 . 08 . Results were normalized to GAPDH and B2M ( GAPDH results shown ) . See also Fig S2C for results in 293T cells . All error bars show SD . ( C ) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups ( day 5 post transductions , or day 2 post 5 - AZA treatment ) . Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group . Mann - Whitney tests were performed to compare shControl to shKAP1 ( p = 0 . 6593 ) and DMSO to 5 - AZA ( 0 . 0027 ) . 5 - AZA was added to all experiments at 7uM . All numbers above bars depict fold changes compared to control cells ( to one decimal place ) .

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Figure 4(A) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following shRNA-mediated KAP1 depletion in HeLa cells (day 6 post transduction), normalized to B2M and GAPDH respectively. n=2 and two-tailed unpaired t test p-values are: HERVK14C: 0.025, 0.003; SVA VNTR: 0.00027, 0.01; ISG56: 0.002; CCL5: 0.007, 0.00003; CXCL10: 0.0008; Mxa: 0.011; IKba: 0.008, 0.01. See also Fig S2AB.(B) qRT-PCR expression of endogenous repeats (left) and ISGs (right) following 5-AZA treatment of HeLa cells and PBMCs (day 6 post transduction). A representative experiment of two is shown in each case. Two-tailed unpaired t tests were performed. HeLa cell p-values: ISG56: 0.005; CCL5: 0.0006; CXCL10: 0.0007; Mxa: 0.0001; IFNb: 0.02. For repeat expression (left panel), p values not available as N=2. PBMC p-values: HERVK14C: 0.1; SVA D VNTR: 0.003; ISG56: 0.001; CCL5: 0.012; CXCL10: 0.012; Mxa: 0.02; IKba: 0.08. Results were normalized to GAPDH and B2M (GAPDH results shown). See also Fig S2C for results in 293T cells. All error bars show SD.(C) DNA methylation at endogenous SVAs was measured over 18 CpGs in the stated treatment groups (day 5 post transductions, or day 2 post 5-AZA treatment). Each point represents the average methylation state of one sequence with at least 10 sequences analyzed per group. Mann-Whitney tests were performed to compare shControl to shKAP1 (p=0.6593) and DMSO to 5-AZA (0.0027). 5-AZA was added to all experiments at 7uM. All numbers above bars depict fold changes compared to control cells (to one decimal place).


Figure 5 ( A ) H3K9me3 ChIP - PCR where error bars show SD of 3 independent IPs . Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region . IgG control IPs gave only background enrichments , shown . ( B ) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1 - depletion ( left ) and ChIP ( right ) . Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate . Results show IP relative to total input and GAPDH normalized . IgG control IPs gave only background enrichments shown . One representative experiment of two is shown . Unpaired t tests were performed . P values : ZNF239 : KAP1 , 0 . 0292 ; SETDB1 , 0 . 0137 ; HERVK14C _ 1 : KAP1 , 0 . 0227 ; SETDB1 , 0 . 0082 ; HERVK14C _ 2 : SETDB1 , 0 . 0462 ; SVA D VNTR , SETDB1 , 0 . 0064 . The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment , normalized to B2M or GAPDH as stated . All error bars show SD . ( C ) Venn diagram showing H3K9me3 enriched sites ( 18271 ) as those identified in both ChIP replicates in the human cell line K562 ( left ) . Right : % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP - seq peak . 5kb upstream and downstream of each gene was also included . N = 437 genes per group . ( D ) An IFIT1 promoter - based Luciferase reporter THP - 1 cell line was used or 293T cells or HeLa cells transduced with an IFN - β promoter - based Luciferase reporter . These cell lines were transduced with an shKAP1 or shControl vector or a vFLIP - expressing vector and luciferase measured 6 days later . Error bars show SD . Unpaired t tests were used to compare shControl to shKAP1 samples : p values = 0 . 122 ( THP - 1 ) , 0 . 0001 ( HeLa ) , 0 . 0001 ( 293T cells ) . ( E ) WT , STING KO and MAVS KO THP - 1 cells were verified by Licor blot . ( F ) Cells from ( E ) were untreated or transduced with shControl or shKAP1 vectors and Luciferase read over time ( left ) . Cells were also treated with control stimulants ( right ) . Results show the average Luciferase secreted over two time points ( days 5 and 6 for the left plot and days 1 and 2 for the right plot ) with all error bars showing SEM . HT - DNA : herring testis DNA .

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Figure 5(A) H3K9me3 ChIP-PCR where error bars show SD of 3 independent IPs. Results show the IP enrichment relative to the total input normalized to GAPDH as a negative control region. IgG control IPs gave only background enrichments, shown.(B) 293T cells were transduced with the stated shRNAs and cells harvested 4 days after puro selection for Western Blot to verify KAP1 and SETDB1-depletion (left) and ChIP (right). Cell numbers were normalized per treatment group before sonication and IPs were performed in triplicate. Results show IP relative to total input and GAPDH normalized. IgG control IPs gave only background enrichments shown. One representative experiment of two is shown. Unpaired t tests were performed. P values: ZNF239: KAP1, 0.0292; SETDB1, 0.0137; HERVK14C_1: KAP1, 0.0227; SETDB1, 0.0082; HERVK14C_2: SETDB1, 0.0462; SVA D VNTR, SETDB1, 0.0064. The inlay shows qRTPCR expression analysis of HERVK14C in the same experiment, normalized to B2M or GAPDH as stated. All error bars show SD.(C) Venn diagram showing H3K9me3 enriched sites (18271) as those identified in both ChIP replicates in the human cell line K562 (left). Right: % of ISGs or random genes intersecting a H3K9me3 or KAP1 ChIP-seq peak. 5kb upstream and downstream of each gene was also included. N=437 genes per group.(D) An IFIT1 promoter-based Luciferase reporter THP-1 cell line was used or 293T cells or HeLa cells transduced with an IFN-β promoter-based Luciferase reporter. These cell lines were transduced with an shKAP1 or shControl vector or a vFLIP-expressing vector and luciferase measured 6 days later. Error bars show SD. Unpaired t tests were used to compare shControl to shKAP1 samples: p values = 0.122 (THP-1), 0.0001 (HeLa), 0.0001 (293T cells).(E) WT, STING KO and MAVS KO THP-1 cells were verified by Licor blot.(F) Cells from (E) were untreated or transduced with shControl or shKAP1 vectors and Luciferase read over time (left). Cells were also treated with control stimulants (right). Results show the average Luciferase secreted over two time points (days 5 and 6 for the left plot and days 1 and 2 for the right plot) with all error bars showing SEM. HT-DNA: herring testis DNA.


Figure 1 ( A ) AP radiograph demonstrates characteristic findings of ATD in the R05 - 365A proband . Note the shortened humeri ( closed arrowhead ) and elongated clavicles ( arrow ) . ( B ) Radiographs of the Cmh001543 - 01 proband showing similar findings .

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Figure 1(A) AP radiograph demonstrates characteristic findings of ATD in the R05-365A proband. Note the shortened humeri (closed arrowhead) and elongated clavicles (arrow). (B) Radiographs of the Cmh001543-01 proband showing similar findings.


Figure 2 ( A ) Sanger sequence trace showing homozygosity for the c . 469C > T mutation , predicting a stop codon in exon 6 of GRK2 ( arrow ) . ( B ) Exome analysis showing the biallelic changes c . 1348 _ 1349del and c . 555 + 1G > A . Red arrow points to the place of mutation . Orange and green boxes indicate the reference nucleotide and its substitution , respectively . ( D ) Expression of GRK2 as measured by RT - PCR in three replicates each of control and R05 - 365A fibroblasts , demonstrating absence of GRK2 transcript in ATD cells . GAPDH served as a positive control . ( E ) Two control and ATD fibroblast samples were immunoblotted for GRK2 . Note the absence of GRK2 protein in the ATD cells . Actin served as a loading control . The data shown are representative of three independent experiments .

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Figure 2(A) Sanger sequence trace showing homozygosity for the c.469C>T mutation, predicting a stop codon in exon 6 of GRK2 (arrow).(B) Exome analysis showing the biallelic changes c. 1348_1349del and c.555 +1G>A. Red arrow points to the place of mutation. Orange and green boxes indicate the reference nucleotide and its substitution, respectively.(D) Expression of GRK2 as measured by RT-PCR in three replicates each of control and R05-365A fibroblasts, demonstrating absence of GRK2 transcript in ATD cells. GAPDH served as a positive control.(E) Two control and ATD fibroblast samples were immunoblotted for GRK2. Note the absence of GRK2 protein in the ATD cells. Actin served as a loading control. The data shown are representative of three independent experiments.


Figure 3 ( A ) GRK2 immunohistochemistry in a control 17 - week gestational age distal femur growth plate demonstrating widespread expression , particularly in the bone ( B ) , proliferating zone of the growth plate ( P ) , and the perichondrium ( arrowheads ) . Non - reactive antibody was used as a control ( Ab control ) . R , reserve zone . H , hypertrophic zone . Scale bars , 100 μm . ( B ) Picrosirius red staining of the femoral growth plates of a control and the ATD R05 - 365A fetus . The control growth plate shows normal architecture with clearly distinguishable reserve ( R ) , proliferating ( P ) and hypertrophic ( H ) zones . A profound disruption of growth plate architecture is obvious in the GRK2 - / - growth plate , which shows irregularly - shaped reserve zone chondrocytes , a lack of discernable proliferating chondrocytes and a significantly under - developed hypertrophic zone . Scale bars , 100 μm . ( C ) Shortened long bones in the chicken wings injected with CMPD101 . The right limb bud was injected with CMPD101 , the left one was left as a control . Note the shortened humerus ( H ) , ulna ( U ) and radius ( R ) in the CMPD101 - injected wing . Mean ± SEM . Mann - Whitney U test ; number of animals is indicated . Scale bar , 150 μm . ( D ) Alcian blue staining of the femoral growth plates of a control and the ATD R05 - 365A fetus . Note the significantly lesser blue staining of the R05 - 365A growth plate stained by alcian blue pH 1 . 0 , suggesting impaired sulfation of the extracellular matrix . Scale bars , 200 μm . ( E ) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101 . Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101 , quantified below . Mean ± SEM . Mann - Whitney U test ; number of micromasses is indicated . A representative experiment of four ( pH 2 . 5 ) and three ( pH 1 . 0 ) is shown . Scale bar , 100 μm . ( F ) Alcian blue staining of the micromasses generated from Grk2 + / + and Grk2 - / - NIH3T3 cells . Note the mildly weaker staining by alcian blue pH 2 . 5 and the nearly absent sulfation stained by alcian blue pH 1 . 0 in the Grk2 - / - micromasses . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of micromasses is indicated . Scale bar , 1 mm .

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Figure 3(A) GRK2 immunohistochemistry in a control 17-week gestational age distal femur growth plate demonstrating widespread expression, particularly in the bone (B), proliferating zone of the growth plate (P), and the perichondrium (arrowheads). Non-reactive antibody was used as a control (Ab control). R, reserve zone. H, hypertrophic zone. Scale bars, 100 μm.(B) Picrosirius red staining of the femoral growth plates of a control and the ATD R05-365A fetus. The control growth plate shows normal architecture with clearly distinguishable reserve (R), proliferating (P) and hypertrophic (H) zones. A profound disruption of growth plate architecture is obvious in the GRK2-/- growth plate, which shows irregularly-shaped reserve zone chondrocytes, a lack of discernable proliferating chondrocytes and a significantly under-developed hypertrophic zone. Scale bars, 100 μm.(C) Shortened long bones in the chicken wings injected with CMPD101. The right limb bud was injected with CMPD101, the left one was left as a control. Note the shortened humerus (H), ulna (U) and radius (R) in the CMPD101-injected wing. Mean ± SEM. Mann-Whitney U test; number of animals is indicated. Scale bar, 150 μm.(D) Alcian blue staining of the femoral growth plates of a control and the ATD R05-365A fetus. Note the significantly lesser blue staining of the R05-365A growth plate stained by alcian blue pH 1.0, suggesting impaired sulfation of the extracellular matrix. Scale bars, 200 μm.(E) Alcian blue staining of the chicken limb bud micromasses treated with GRK2 inhibitor CMPD101. Note the gradual inhibition of the cartilage nodules with increasing concentration of CMPD101, quantified below. Mean ± SEM. Mann-Whitney U test; number of micromasses is indicated. A representative experiment of four (pH 2.5) and three (pH 1.0) is shown. Scale bar, 100 μm. (F) Alcian blue staining of the micromasses generated from Grk2+/+ and Grk2-/- NIH3T3 cells. Note the mildly weaker staining by alcian blue pH 2.5 and the nearly absent sulfation stained by alcian blue pH 1.0 in the Grk2-/- micromasses. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of micromasses is indicated. Scale bar, 1 mm.


Figure 4 ( A , B ) Defective GLI3 processing in GRK2 - / - fibroblasts . Cells were serum - starved to induce cilia before treatment with the Hh pathway activator SAG ( Smoothened agonist ) , and the whole cell lysates were immunoblotted for GLI3 . Note the SAG - mediated downregulation of both full - length ( FL ) and repressor ( R ) GLI3 in control fibroblasts , demonstrating normal processing . There was no effect of SAG on GLI3 processing in GRK2 - / - cells . A representative experiment of four is shown . ( B ) Quantitation of the GLI3FL to GLI3R ratio using densitometry . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated . ( C ) Defective SAG response in GRK2 - / - fibroblasts . Cells were treated with SAG for 24 hours . Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT - PCR ; GAPDH was used for normalization . Note the SAG - mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2 - / - cells . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated . ( D ) Under - phosphorylation of Smoothened ( SMO ) in GRK2 - / - fibroblasts . Lysates of SMO - transfected cells were resolved by the phospho ( p ) - shift PAGE and immunoblotted for SMO . The portion of pSMO analyzed by densitometry is shown . GRK2 add - back demonstrates a rescue of the pSMO in GRK2 - / - fibroblasts . Mean ± SEM . Mann - Whitney U test ; number of biological replicates is indicated . Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . Cells were treated with SAG and immunostained for SMO , ARL13B and pericentrin . Note the failure in SMO accumulation in cilia of GRK2 - / - cells ( arrow ) . Scale bar , 2 µm ( E ) . Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . The percentage of SMO - positive cilia was calculated and plotted . Mean ± SEM . Welch ´ s t - test ; number of biological experiments and the total numbers of analyzed cilia are indicated ( F ) . Defective SMO accumulation in cilia of GRK2 - / - fibroblasts . The intensity of ciliary SMO was analyzed and plotted . Central band , median . Box , 1st - 3rd quartile . Whiskers , 10 % - 90 % percentile . Mann - Whitney U test ; number of biological experiments and the total numbers of analyzed cilia are indicated ( G ) .

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Figure 4(A,B) Defective GLI3 processing in GRK2-/- fibroblasts. Cells were serum-starved to induce cilia before treatment with the Hh pathway activator SAG (Smoothened agonist), and the whole cell lysates were immunoblotted for GLI3. Note the SAG-mediated downregulation of both full-length (FL) and repressor (R) GLI3 in control fibroblasts, demonstrating normal processing. There was no effect of SAG on GLI3 processing in GRK2-/- cells. A representative experiment of four is shown. (B) Quantitation of the GLI3FL to GLI3R ratio using densitometry. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.(C) Defective SAG response in GRK2-/- fibroblasts. Cells were treated with SAG for 24 hours. Transcript levels of GLI3 targets GLI1 and PTCH1 were determined by qRT-PCR; GAPDH was used for normalization. Note the SAG-mediated induction of GLI1 and PTCH1 expression in controls but not in GRK2-/- cells. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.(D) Under-phosphorylation of Smoothened (SMO) in GRK2-/- fibroblasts. Lysates of SMO-transfected cells were resolved by the phospho(p)-shift PAGE and immunoblotted for SMO. The portion of pSMO analyzed by densitometry is shown. GRK2 add-back demonstrates a rescue of the pSMO in GRK2-/- fibroblasts. Mean ± SEM. Mann-Whitney U test; number of biological replicates is indicated.Defective SMO accumulation in cilia of GRK2-/- fibroblasts. Cells were treated with SAG and immunostained for SMO, ARL13B and pericentrin. Note the failure in SMO accumulation in cilia of GRK2-/- cells (arrow). Scale bar, 2 µm (E).Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The percentage of SMO-positive cilia was calculated and plotted. Mean ± SEM. Welch´s t-test; number of biological experiments and the total numbers of analyzed cilia are indicated (F).Defective SMO accumulation in cilia of GRK2-/- fibroblasts. The intensity of ciliary SMO was analyzed and plotted. Central band, median. Box, 1st-3rd quartile. Whiskers, 10%-90% percentile. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated (G).