(A) USF-1 immunoprecipitates using monoclonal anti-USF-1 antibodies was Western blotted with polyclonal anti-USF-1 or anti-P-USF-1 antibodies. Immunoblotting with anti-P-USF-1 in the presence of peptide or with pre-immune serum are shown as controls. (B) ChIP for indicated proteins binding to the −444 FAS-CAT promoter in mouse liver. (C) ChIP using anti-FLAG antibodies (top) for WT USF-1 and S262 USF-1 mutant association to the FAS promoter in 293FT cells. The FAS promoter activity (bottom) in cells transfected with −444 FAS-Luc and WT USF-1 or S262 USF-1 mutants was monitored. Immunoblotting for protein levels of WT, S262 USF-1 mutants (insert) and FAS are shown. (D) Immunoprecipitated USF-1 was Western blotted with indicated antibodies. (E) ChIP for binding of indicated proteins to the −444 FAS-CAT promoter in mouse liver. (F) ChIP (top) for association of WT USF-1 and K237 USF-1 mutant to the FAS promoter in 293F cells. The promoter activity of the −444 FAS-Luc (bottom) in cells transfected with WT USF-1 or K237 USF-1 mutants was measured.

(A) IP of cells overexpressing USF-1 and P/CAF with FLAG antibodies (top). Bacterially expressed USF-1 was in vitro acetylated with P/CAF (bottom). (B) IP of WT USF-1 or K237A USF-1 from cells transfected with P/CAF using FLAG antibodies (top left). P/CAF protein levels were analyzed by immunoblotting. IP of cells transfected with USF-1 and P/CAF (top right) or cells transfected with P/CAF along with WT USF-1 or USF-1 mutants (bottom). (C) IP of cells overexpressing USF-1 and P/CAF with HDAC9 using FLAG antibodies. (D) Bacterially expressed USF-1-GST was incubated with in vitro translated ³⁵S-labeled P/CAF and HDAC9 before subjecting to GST-pull down. Purified USF-1 was subjected to autoradiography for labeled proteins. GST alone was used as a control. (E) The promoter activity of the -444 FAS-Luc upon cotransfection of USF-1 along with P/CAF or HDAC9. (F) The promoter activity of −444 FAS-Luc upon cotransfection of USF-1 along with increasing amount of P/CAF or HDAC9 was measured. Total cell lysates were immunoblotted for indicated proteins.

(A) IP of FLAG-tagged USF-1 from cells transfected with DNA-PK using FLAG antibodies. Empty vector was used as control. (B) IP with FLAG antibodies of USF-1-FLAG from DMSO or OA treated cells overexpressing USF-1 (left) and from cells cotransfected with USF-1 and control or PP1 siRNA (right). (C) IP with FLAG antibodies of 293F cells transfected with FLAG-tagged WT USF-1 or S262 USF-1 mutants. Nuclear extracts from non-transfected cells were used as a control. (D) IP of USF-1-FLAG from HepG2 cells transfected with FLAG-tagged S262 USF-1 mutants. Immunoprecipitated USF-1 was immunoblotted with indicated antibodies (top). Total protein levels were also analyzed by immnnoblotting (bottom). (E) IP of nuclear extracts from HepG2 cells transfected with control or DNA-PK siRNA using monoclonal anti-USF-1 antibodies. (F) IP of nuclear extracts from M059J or M059K cells using anti-USF-1 antibodies. (G) ChIP for binding of indicated proteins to the FAS promoter in M059J or M059K cells. (H) IP of liver nuclear extracts from WT and SCID mice using monoclonal anti-USF-1 antibodies. (I) ChIP for indicated protein association to the FAS promoter in mouse liver. ChIP samples were analyzed by semi-quantitative PCR (top) or qPCR (bottom). (J) IP of liver nuclear extracts using anti-USF-1 antibodies. (K) ChIP for indicated protein association to the FAS promoter in liver.

(A) Nascent RNA from liver nuclei were used for RT-PCR. (B) Nascent RNA from liver nuclei were used for RT-qPCR. Fold induction normalized by β-actin was shown. (C) Run-on of biotin labeled nascent transcripts from liver nuclei at different time points upon feeding were analyzed by RT-qPCR. (D) ChIP for biotin incorporation and indicated protein binding to the FAS promoter in liver. (E) Newly synthesized fatty acids during a 4 hr-period of body water labeling with ²H₂O in livers from 9-wk old WT and SCID mice during feeding were measured. Values are means ± SEM, n = 12. (F) Equal amounts of Liver extracts from 9-wk old WT and SCID mice after 24 hrs of feeding were immunoblotted for total FAS protein levels. (G) Hepatic and serum triglyceride levels were measured in 9-wk old fed WT and SCID mice. (H) Schematic representation of USF-1 and its interacting partners and their effects on lipogenic gene transcription in fasting/feeding.

(A) Bacterially expressed USF-1 was incubated with DNA-PK in the presence or absence of ATP or wortmannin (2 uM). (B) IP of cells overexpressing WT USF-1 and DNA-PK, T3950D DNA-PK (kinase dead) (left), T3950A DNA-PK or empty vector (middle) or overexpressing S262A USF-1 mutant with or without DNA-PK (right), using FLAG antibodies. DNA-PK protein levels were analyzed by immnoblotting. (C) IP (left) of cells cotransfected with USF-1, control siRNA or DNA-PK siRNA. The promoter activity of −444 FAS-Luc in cells transfected with WT USF-1 or siRNA was measured (right). (D) DNA-PK activity in liver nuclear extracts pretreated with or without wortmannin (2uM) was assayed using[ γ³²P] ATP and biotinylated p53 peptide as substrate. (E) IP of liver nuclear extracts (top) or nuclear extracts from HepG2 cells (bottom) using anti-DNA-PK antibodies. (F) Total and phosphorylated DNA-PK levels were analyzed by westernblotting. IP of USF-1-FLAG from cells overexpressing USF-1 treated with either control DMSO or OA at 1uM (left) and Taut at indicated concentrations for 2 hrs (right). DNA-PK phosphorylation was detected from cells treated with Taut at 1uM for 2hrs. (G) IP of cells cotransfected with USF-1 and control or PP1 siRNA with anti-USF-1 antibodies. PP1 protein levels were analyzed by Western blotting as shown. (H) IP of nuclear extracts or total lysates from livers (left) or HepG2 cells (right) with anti-PP1 antibodies. USF-1 and β-actin protein levels were analyzed by Western blotting.

(A) The identities (left) of USF-1-associated polypeptides analyzed by MS are listed. Purified USF-1 interacting proteins were separated by SDS-PAGE for silver staining (2^nd left). Immunoblotting of TAP eluates (middle) using antibodies against indicated proteins. Immunoprecipitated USF-1 (2^nd left) from 293F cells with monoclonal anti-USF-1 antibodies was Western blotted with indicated antibodies. TAP eluates from 293F cells transfected with USF-1-TAP and HDAC9-FLAG were immunoblotted with indicated antibodies (right). (B) RNA from mouse liver and adipose tissue were used for RT-PCR. (C) ChIP for association of USF-1 interacting proteins to the −444 FAS-CAT promoter (left) or the mGPAT promoter (right) in livers from fasted or fed FAS-CAT transgenic mice (left). (D) FAS and mGPAT expression in liver determined by RT-qPCR. (E) IP of FLAG-tagged USF-1 from HepG2 cells treated with or without 100 nM insulin for 30 min. Immunoprecipitated USF-1 was immunoblotted with indicated antibodies. (F) ChIP for association of USF-1 interacting proteins to the −444(−65m) FAS-CAT (left) promoter in liver or the FAS promoter in HepG2 cells (right) transfected with control or USF-1 siRNA. USF-1 protein levels were analyzed by immunoblotting (bottom right). (G) ChIP for binding of USF-1 interacting proteins to the −444 FAS-CAT (left) and −444(−150m) FAS-CAT (right) promoter regions in mouse liver. (H) ChIP analysis for biotin incorporation into 3'-ends of DNA breaks and DNA-PK and TopoIIβ binding to the FAS-CAT (left) or the endogenous FAS promoter (right) in livers from fasted or fed FAS-CAT transgenic or wild type mice.