Mechanisms of activation
Based on the structural characteristics of nuclear receptors, their activity can be controlled via three different mechanisms:
Firstly, the classic way is that ligands induce the recruitment of coregulators. Most often, in the absence of ligand, the steroid receptors (such as AR, GR) are located in the cytoplasm and associated with heat shock proteins; upon steroid hormone binding, receptors dissociate from the complex, dimerize, translocate into the nucleus, bind to the response elements on their target genes, and recruit specific coactivators to activate gene expression. Non-steroid receptors are in the nucleus and bound to histone deacetylases which keep the chromatin in a repressive state, ligand binding induces corepressor dissociation which facilitates interaction of the receptors with chromatin remodelers and modifiers, such as SRA, p/CAF, CBP/P300, finally leading to de-repression (Giguère, 1999; Rochette-Egly, 2003; Tecalco-Cruz, 2018) (Figure 3).
Figure 6. Positive (red arrows) and negative (green arrows) effects of NR phosphorylation on the transactivation of target genes. Adapted from (Rochette-Egly, 2003).
The switch ability of nuclear receptors between activation and repression in response to environmental signals is mainly ascribed to the recruitment of numerous and diverse coregulators (coactivators and corepressors). Up to now, there are about 300 coregulators
identified in human Lo a d a d O Malle , . Evidences have shown that coregulators work as multicomponent protein complexes and are subjected to dynamic rearrangements which are required for specific transcription (Kato et al., 2011).
As the name suggests, coactivators can be directly recruited by NRs and promote NR-target genes expression, the common structural feature of NRs coactivators is an LxxLL motif (NR box) which recognizes and binds the AF-2 domain (Kato et al., 2011). A largely shared functional property of coactivators is acetyltransferase activity which looses chromatin and facilitates the recruitment of other chromatin modifiers. The first identified coactivators were the members of the SRC/p160 family (SRC-1/NCoA-1, SRC-2/GRIP-1/TIF2 and SRC-3/CIP/RAC3) (Hong et al., 1996; Li et al., 1997; Oñate et al., 1995). Studies have shown that SRC proteins build a complex containing approximately 6-10 stably associated proteins and a large number of less tightly bound ones. These complexes contain enzymatic proteins, such as acetyltransferase CBP/p300, ubiquitin ligases such as E6-AP, the ATP-dependent chromatin remodeling SWI/SNF complex, the RNA coactivator SRA, the protein methyltransferases CARM-1 and PRMT-1 (Chakravarti et al., 1996; Chen et al., 1999b; Lanz et al., 1999; Nawaz et al., 1999; Yoshinaga et al., 1992). Mechanistically, enzymatic proteins can exert covalent modifications (including phosphorylation, acetylation, sumoylation, ubiquitination) both on the coactivators and NRs, the PTMs can enhance the coactivator enzymatic activities to promote the recruitment of other coregulators or dismantling the inhibitory ones (Kato et al., 2011). In addition, cellular signaling pathway kinases also make a contribution to the NRs or coactivators modifications. For instance, SRC-3 can be activated through a GSK3-mediated phosphorylation-mono-ubiquitination. After a transcription round, SRC-3 undergoes degradation through poly-ubiquitination (Wu et al., 2007); SRC-3 can also be targeted by other modifications (methylation and acetylation can dismantle the coregulatory complex and contribute to reorganize other complexes) to form divergent multiprotein complexes to impact distinct functions of NRs M Ke a a d O Malle , .
In summary, ligand-specified variations in the AF-2 domain, along with covalent modifications and signaling cascade kinases can have a huge difference on the capacity of a receptor to recruit coactivators, leading to the production of a variety of genes expression and pervasive biological consequences M Ke a a d O Malle , ; ‘ose feld et al.,
The negative counterparts of coactivators are corepressors which o tai s Co‘N‘ o . They can be recruited by NRs in the absence of ligand or in the presence of NR synthetic antagonists such as Tamoxifen M Ke a a d O Malle , .Corepressors include NCoR and SMRT (identified through biochemical purification) which recruit HDAC3, GPS2 and the transducing-like factors TBL1 and TBLR1, with HDAC3 deacetylase activity being essential for the repression (Pérez-Schindler et al., 2012;Webb et al., 2000;Yoon et al., 2003).Their function is comparable with coactivator that is they are not a bridge between the environment and NRs but instead as enzymatic tools which are necessary to the transcription. Besides, the PTMs placing on corepressors influence gene expression targeted by transcription factors (like NRs) with which the corepressor interacts (Rosenfeld et al., 2006). For instance, phosphorylation of NCoR can lead to its relocalization to the cytoplasm and reduce mRNA production in nucleus, while the sumoylation can enhance its corepressor capacity (Jonas and Privalsky, 2004; Tiefenbach et al., 2006), thus the combination among PTMs, coregulators, and NRs produce distinct transcriptional effects (Kato et al., 2011; Lo a d a d O Malle , ;).
NRs and disease
Nuclear receptors regulate genetic network through sensing the environmental cues, including steroids, retinoid, fatty acid, lipids… Based on the results obtained with knock-out mice and functional mutations studies, NRs have been shown as involved in embryonic development, metabolic pathways, reproductive and cell growth. In another word, if NRs are misregulated, they may cause diverse diseases, ranging from obesity, hypertension, cardiovascular problems to cancer. A few examples will illustrate this view (Bushue and Wan, 2010; Gkikas et al., 2017; Sun and Shi, 2010; Xie, 2010).
The LXR subfamily comprises two sub-t pes, LX‘α a d LX‘β, a d is highly expressed in liver, kidney and intestine. Both LXRs bind to DNA with RXRs to regulate transcription. Studies have shown that one of the LXRs direct target is ABCA1 which is required for the process of reverse cholesterol; loss of functional ABCA1 results in Tangier disease which present large accumulation of cholesterol-laden macrophages and constitutes a risk for the development of atherosclerosis (Costet et al., 2000; Hayden et al., 2000). In addition, ABCA1 defective cells cannot efflux cholesterol. Treating primary macrophages cell lines with LXR agonist can induce the expression of ABCA1 and increase cholesterol efflux, supporting the role of LXR as a regulator of ABCA1 and its inhibitory effect on the progression of atherosclerosis. This is consistent with the results obtained in Lxrα-/-/Lxrβ-/- mice, showing accumulation of cholesterol-laden macrophages and increased atherosclerosis (Wagner et al., 2003). Moreover, activation of LXRs represses the expression of genes encoding enzymes of gluconeogenesis in the liver, induces the expression of GLUT4 in adipose tissue, decreases hepatic glucose output and blood glucose levels in animal model of type II diabetes, all the activities of activation LXRs mimic the treatment with insulin, suggesting another role for LXRs as a regulator in metabolism (Cao et al., 2003; Laffitte et al., 2003; Schulman, 2010).
The androgen receptor (AR) plays pivotal roles in the development of the prostate gland, it is highly expressed in reproductive tissues such as the prostate, the adrenal gland and the epididymis (Keller et al., 1996). Studies showed that AR is a therapeutic target in prostate cancer. Based on the classic structure and genomic mechanism pathway, the expression of AR target gene relies on interaction with coactivators in LBD induced by androgen binding. The majority of therapeutic strategies are focused on LBD-dependent mechanism to modulate AR function (McEwan, 2004). Up to now, quite a lot of AR-binding molecules have been designed, including agonists and antagonists. AR agonists are mainly used to maintain secondary sex characteristics, and a useful agent is testosterone, but the dose should be monitored carefully, since excessive testosterone can cause erythrocytosis and other side effects, such as virilizing or feminizing; moreover, this androgen is not orally available due to metabolic instability (Bhasin et al., 2001). A great deal of attention is focused on nonsteroidal ligands, aiming to identify tissue selective, orally bioavailable AR ligands (Gao, 2010). AR antagonists are mainly used in prostate diseases, and most of antagonist mechanism is through inhibiting androgen binding to AR. However, these ligands cannot function in case of AR mutations (in which testosterone binding site is changed or in which antagonist can activate mutated AR) nor in advanced stages of disease (where AR acts in an androgen-independent manner) (Brinkmann and Trapman, 2000). The strategy is now focused on novel antagonists which block AR function in functional domain, like AF2 region, (Gao, 2010).
In summary, nuclear receptors regulate a wide range of biological processes, they are essential for normal development and are also good therapeutic targets for the treatment of cancer or metabolic diseases. However, many ligands are limited in clinical treatment due to a number of side effects. Crystal structure studies revealed insights into the mechanisms of ligand binding affinity and specificity, as well as differential recruitment of coregulators. Structure-based drug design will be a promising therapeutic approach (Chen, 2010; Jin and Li, 2010).
Table of contents :
Chapter I –Nuclear Receptors: general concepts
1.1 Basic structure and molecular functions of nuclear receptors
1.2 Mechanisms of activation
1.3 NRs coregulators
1.4 NRs and disease
2.1 Structural and expression pattern
2.4 Action mechanism
2.4.1 PGC-1 coactivators
2.5 Post-translational modifications
2.6 Physiological functions
3.1 The role of Epigenetics
3.2 Histone modification
3.3 Histone modification mechanism in transcription
3.4 Histone methylation
3.5 Methylation and disease
Chapter IV–Histone lysine demethylation
4.1 Histone lysine specific demethylase 1 ( LSD1)
4.2 LSD1 represses gene expression
4.2.1 With BHC and CoREST
4.2.2 With NuRD
4.3 LSD1 activates gene expression
4.4 LSD1 demethylates more than histone
4.5 Biological effects of LSD1
4.6 LSD1 and metabolism
Chapter V–Nuclear Respiratory Factor 1 (NRF1)
5.1 General introduction
5.2 NRF1 and disease
Chapter VI–Histone methyltransferases (HTMs)
6.1 Identification of SET7 and its structural analysis
6.2 SET7 mediates H3K4 mono-methylation
6.3 Physiological role of H3K4 methylation
6.4 Non-histone lysine methylation
6.5 Examples of SET7 mediated non-histone methylation and biological effects
6.5.1 SET7-mediated lysine methylation and protein stability
6.5.2 SET7-mediated lysine methylation and transcriptional activity
6.5.3 SET7-mediated lysine methylation and cellular location
6.6 SET7 involvement in disease
Chapter VII— ETS family
7.1 General introduction of ETS family and structure
7.2 Biological roles
7.3.1 Structure of human ETS1
7.3.2 Transcriptional regulation
7.3.3 Physiological role of ETS1
General introduction of article 1
General introduction of article 2
General Introduction of project 3
Discussion and perspective
Common aspects between both two complexes.
RNA-seq information and Oligonucleotides
Table 1. SET7-modulated genes
Table 2. Genes modulated by siSET7 and siERR
Table 3. Genes modulated by siSET7, but not by siERR.
Table 4. Genes modulated by siERR, but not by siSET7.
Table 5. Oligonucleotides used in this study
Articles published as first author