Specific Aims
1. Generation and validation of coregulator knockdown reagents in adipocytes and mouse liver.
Targeting siRNAs will be validated first in cell culture, then subcloned into lentiviruses with the ability to infect mature adipocytes in culture as well as hepatic parenchyma in vivo. The lentivirus backbone will be identical to that used in other Bridging Projects, to allow cross-comparison. The effectiveness of the lentiviral approaches in adipocytes and liver will be confirmed by Q-PCR and Western analysis.
2. Determine the extent and integration of corepressor regulation of gene expression in adipocytes.
The effect of knockdown of corepressors (N-CoR, SMRT, RIP140) as well as Class I HDACs which they recruit (HDACs 1, 2, and 3) on gene expression in 3T3-L1 preadipocytes as well as mature adipocytes will be assessed by infection of lentivirus expressing cognate coregulator siRNAs, validation of knockdown, preparation of RNA, and gene expression analysis using 44K Affymetrix oligonucleotide arrays. Datasets will be analyzed in collaboration with the Bioinformatics Resource using Ingenuity and Vampire pathway analysis software. Analysis will also be performed across corepressor/HDAC knockdowns, to interrogate coregulation or opposing regulation of identical pathways, and will compare corepressor/HDAC knockdown to knockdown of individual NRs, using bioinformatics approaches to link specific NRs to specific corepressor pathways. In collaboration with other Bridging Projects in this Strand (Evans, Mangelsdorf, and Moore), QPCR analysis will also be performed with focus on quantitative analysis of NRs and coregulators, as well as ABC transporters, cytochrome P450 enzymes, and micro RNAs.
3. Determine the extent and integration of corepressor regulation of gene expression in mouse liver.
The effect of corepressor and HDAC knockdown on gene expression in mouse liver will be assessed by tail vein injection of lentivirus that expresses cognate coregulator siRNA. Validation of knockdown will be assessed by mRNA, Western, and immunohistochemistry. Global gene expression changes will be analyzed using 44K Affymetrix oligonucleotide arrays, and datasets will be analyzed using pathway analysis software. Analysis will also be performed across corepressor/HDAC knockdowns, to interrogate coregulation or opposing regulation of identical pathways, and will compare corpressor/HDAC knockdown to knockdown of individual NRs, using bioinformatic to link specific NRs to specific repression pathways. QPCR analysis will also be performed with focus on quantitative analysis of NRs and coregulators, as well as ABC transporters, cytochrome P450 enzymes, and micro RNAs.
