PPARα inhibits progression of steatohepatitis to fibrosis via a DNA

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DNA RESPONSE ELEMENTS GEOMETRY, ARCHITECTURE AND RECOGNITION BY NUCLEAR RECEPTORS

DNA sequence recognition and binding is the initial step of the transactivation process mediated by nuclear receptors. Consequently, NR monomers or dimers are positioned on RE which are made of one or two hexameric half-site motifs. Adopting a different geometry, they form palindromes, direct (DR), everted (ER) or inverted repeats (IR) separated by a spacer of varying length and sequence. Four conditions can be distinguished that determine the uniqueness of the response element. They are (i) the nucleotide sequence of the DNA-half sites, (ii) their relative orientation (iii) the sequence of the spacer and (iv) the length of the spacer.
Some NRs, mainly orphans, bind to DNA as monomers. The monomeric Nurr1 binds to a hormone response element 5′-AGGTCA-3′ flanked by a 5′ 1 to 6-bp long A/T-rich sequence [142]. This sequence referred to as an Nur77/NGFI-B response element (NBRE) [143] is also the target of Nur77 monomers [144]. Nurr1 can however dimerize with RXR, and in this configuration can display significant affinity for DR with spacing ranging from 10 to 27 bases [145]. A similar promiscuity in binding to naked DNA is observed for SF-1, FTZ-F1, rev-Erb-α and RORα which target a single copy of this extended core recognition sequence, although rev-Erb-α can also bind to a specific DR-2 RE [146,147].

GENERAL MECHANISMS OF TRANSCRIPTIONAL REGULATION BY NUCLEAR RECEPTORS

As already mentioned above, nuclear receptors can control transcriptional events by exerting either a positive, direct effect or by imposing a repressed state to regulated promoters. They can also mediate, through protein-protein interaction, a repressive effect on a variety of other signaling pathways under the control of transcription factors such as AP-1, NF-kappa-B or C/EBP. Each of these aspects will be described below to provide a global view of the most recent concepts which have emerged in the field in the past years (Figure 4).

Nuclear receptor corepressor binding

In the unliganded state, NRs are associated to corepressor complexes. These complexes are composed of a subunit (SMRT/NCoR2 or NCoR1) directly interacting with the receptor through a degenerated LXXLL motif, which harbor a consensus sequence L/I-X-X-I/V-I or LXXXI/LXXXI/L also called the CoRNR box [176,177]. This CoRNR box motif interacts, as the coactivator LXXLL motif, with amino acids from the LBD hydrophobic groove. This interaction interface is remodeled upon agonist binding and helix 12 positioning occludes part of the CoR binding interface. As mentioned above, additional CoR binding interfaces, as well as novel CoRNR boxes have been described [118,119,178], suggesting the use of alternative mechanisms for NR-corepressor interaction. Corepressor complexes are built around the SMRT or NCoR subunits, which harbor a conserved repression domain on which the core repressive machinery (including HDAC3, GPS2 and TBL1 or TBLR1) is assembled. Recent structural and functional studies highlighted a central role for TBL1 in assembling this very large complex (ca. 1-2 MDa) [179]. In some cases, ligand-binding is sufficient to inhibit co-repressor recruitment (e.g. for RXR and TR), but more generally the active removal of the co-repressor complex is required. This points again to the critical role of TBL1/TBLR1 which encompass a F-box domain interacting with the ubiquitin-conjugating enzyme H5 (UBCH5) and a 19S-proteasome complex, which mediates ubiquitination and proteosomal degradation of SMRT- or NCoR-GPS2-HDAC3 complexes [180].

Nuclear receptor coactivator binding

Since the seminal discovery of SRC-1/NCoA1 as a progesterone receptor coactivator [181], more than 350 coactivators have been identified so far. This prodigious amount of polypeptides exhibit various enzymatic activities involved in the regulation of histone modification and chromatin remodeling, initiation of transcription, elongation of RNA transcripts, mRNA splicing and elongation, and proteasomal termination of nuclear receptor complexes. Their involvement and relative activity in nuclear receptor-controlled processes is modulated by their cell-specific expression levels and post-translational modifications, conditions which have been reviewed recently [182,183]. It is also nowadays accepted tha many of these coregulators participate in molecular events driven by other transcription factors.
The coactivator family has been divided in two subfamilies. The first one defines coactivators which interact directly with NR AF-1&2 regions such as the SRC coactivators, CBP and p300. The second one includes other proteins which interact with primary coactivators such as CARM1, CoCoA, Fli-I… Primary and secondary coactivators are recruited to regulated promoters in an orchestrated fashion [184]. Since this issue is devoted to nuclear receptors involved in metabolism control, only coactivators associated to such an activity will be briefly described here.
The p160 and p300 families: Co-activators belonging to the p160 family [NCoA1/SRC-1, NCoA2/TIF2 (known as SRC-2 or GRIP1) and NCoA3/RAC3 (also known as SRC-3, ACTR, pCIP or TRAM-1)], p300 and the cAMP response element-binding protein (CBP) bind to the NR LBD via an alpha-helical LXXLL motif [185,186]. Co-activators such as CBP and p300 posses histone acetylase transferase (HAT) activity, which has a critical role in regulating NR-mediated transcription [187]. N-terminal tail acetylation of histone H4, which is likely to establish contacts with the histone H2A/H2B dimer, prevents this interaction and destabilizes chromatin compaction. Additionally, acetylation weakens the interaction of the histone tails with DNA [188]. Consequently, the chromatin is decondensated allowing the promoter initiation complex to bind at the promoter site.
Data emerging from studies of knockout animals suggest that the SRCs play critical and distinct roles in controlling energy homeostasis. SRC-1-/- mice have decreased energy expenditure and are prone to obesity. In opposition, SRC-2-/- mice are protected against high-fat diet-induced obesity, but can lead to a condition reminiscent of a glycogen storage disease type 1a. The ablation of SRC-3 generates mice highly resistant to high-fat diet-induced obesity. Collectively, these data and others point to a complex, but critical role of SRCs in metabolic regulation which has been in most instances related to the control of PPARγ transcriptional activity [189]. However, given the pleiotropic role of SRCs, it is very likely that other mechanisms contribute to these metabolic effects.

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The ATP-dependent remodelling complex SWI/SNF:

the SWI/SNF complex has a role in metabolic control, as it was identified in yeast to be essential for mating-type switching and growth on sucrose. The SWI/SNF family is evolutionary conserved and plays an important role in ATP-dependent chromatin remodeling [190] by catalyzing the disruption of DNA-histone interactions and sliding of the nucleosome along DNA [191]. The human homolog BAF complex is a multimeric entity of 1.2 MDa including BRG1/hBRM, BAF polypeptides (BAF155/170, BAF60, BAF57, BAF53a/b, BAF47, BAF250a/b, BAF200, BAF45a/b/c/ d, Brd9, and Brd7) and actin. Several of these subunits harbor LXXLL motifs and have been identified not only as nuclear receptors coactivators for ER [192,193], AR [194], RAR [193,195], FXR [196] and GR [197], but also as corepressors of SHP [198], as SWI/SNF components can be integrated in corepressor complexes [199]. Interestingly, the BAF60a subunit displays a circadian expression in mouse liver and, acting as a coregulator of RORα, regulates the expression of clock and metabolic genes [200].

Table of contents :

Chapter 1 General introduction p
Chapter 2 General molecular biology and architecture of nuclear receptors.
Curr Top Med Chem. 2012;12(6):486-504. Review.
Chapter 3 Molecular mechanism of PPARα action and its impact on lipid
metabolism and inflammation.
Advances in Experimental Medicine and Biology: Systems Biology
and Molecular Genetics of PPARs. Springer, New York. 2013.Book
Chapter 4 Approaches used in the thesis
Chapter 5 PPARα inhibits progression of steatohepatitis to fibrosis via a DNA
binding-independent mechanism
Submitted – positive reviewing
Chapter 6 LRH-1 plays a central role in hepatic triglyceride metabolism
Submitted
Chapter 7 Summary and perspectives
List of abbreviations

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