**3.2 Isoform specific transcriptional regulation by members of the ERR family of transcriptional factors**

The ERR subfamily of nuclear receptors comprises three members: ERRα (NR3B1), ERRβ (NR3B2), and ERRγ (NR3B3), with all members having high amino acid sequence homology. ERRβ and ERRγ have high conservations in their LBD domain, where they share less similarities with ERRα. A distinct difference in ERRα is the presence of a phenylalanine at F382 that significantly alters the size and shape of the ligand binding pocket. As a result, ERRα cannot bind to 4-hydroxytamoxifen which acts as antagonist for ERRβ, γ as well as ERs. ERRα, the first orphan nuclear receptor identified from its close homology to Erα [26], is ubiquitously expressed in all cells and tissues, and highly expressed in high oxidative organs. In these tissues/ cells, ERRα regulates the expression of genes involved in glycolysis, such as glyceraldehyde dehydrogenase (GAPDH), and binds to the glucose transporter family members [27]. In breast cancer cells, decreased uptake of glucose is observed in the absence of ERRα [28]. Therefore, ERRα is recognized as an important transcriptional activator for cellular glucose metabolism in response to environmental stimuli [29].

In addition, ERRα is identified as the key transcriptional factor for the regulation of OXPHOS [19–22]. Using mouse myocytes to screen for cis-regulatory elements responsible for the regulation of OXPHOS by PCG-1α and β (Peroxisomal proliferation activated receptor γ (PPARγ) coactivator-1), ERRα was identified together with a ETS family of transcription regulators. In this study, 20 common motifs are identified from over 5000 differentially expressed genes induced by the exogenous expression of PGC-1α (1–3 days) [20]. A majority of the 20 motifs, particularly those that displayed changes early in days 1 and 2, are related to ERR regulated promoters. The ERR motifs are found in >50% of the OXPHOS genes coregulated by PGC-1α. Supporting this analysis, a study performed in livers from type 2 diabetes patients identified ERRα and PPARγ as the two nuclear factors correlated with OXPHOS and can be used as predictors for fasting glucose levels [19]. Adenovirus-mediated expression of PGC-1α in ERRα positive and negative mouse embryonic fibroblasts (mEFs) derived showed that genes regulating mitochondrial functions were among the primary transcripts differentially regulated when ERRα is lost [30]. In particular, the inability of PGC-1α to induce citrate synthase activity, a key indicator of mitochondrial activity in the absence of ERRα supports the role of ERRα in the regulation of mitochondrial function.

ERRα regulates the expression of genes that form the mitochondrial respiratory system, including those that encode proteins involved in the TCA mitochondrial oxidative phosphorylation, respiratory chain, and TCA cycle (**Figure 2**). In addition to regulating the expression of genes encoding mitochondrial proteins, inhibition of ERRα also diminishes the ability of PGC-1α to increase mitochondrial DNA content [21]. In SAOS2 cells where PGC-1α induces mitochondrial biogenesis and function, ERRα is needed for sustaining the expression of TFAM, a mitochondrial transcriptional factor that induces mitochondrial DNA replication and transcription, Tim22, a core translocase protein responsible for the integrity of mitochondrial inner membrane proteins, isocitrate dehydrogenase α, which catalyzes the irreversible oxidative decarboxylation of isocitrate to yield α-ketoglutarate (α-KG) and CO2 as part of the TCA cycle, carnitine/acylcarnitine translocase, the rate limiting enzyme for fatty

*Transcriptional Regulation by ERR and Its Role in NAFLD Pathogenesis DOI: http://dx.doi.org/10.5772/intechopen.109089*

### **Figure 2.**

*ERR is a master regulator for mitochondrial functions and biogenesis. ERR regulates the gene expression of metabolic enzymes and mitochondrial respiratory complexes in the nucleus and mitochondria. PGC-1α binds to NR3B as a coactivator, entering subsequent transcriptional process. It further impacts transcriptional factors such as NRF-1/2, TFAM, POLRMT, TFB1/2M, METERF. Mitochondrial DNA (mtDNA) transcriptional activity is then activated. Meanwhile, genes encode proteins involved in mitochondrial oxidative phosphorylation, respiratory chain, and TCA cycle, including cytochrome C (Cytc), NADH dehydrogenase are regulated as well.*

acid oxidation, as well as Cytochrome *c* and ATP synβ, both directly involved in the electron transportation during OXPHOS [21].

The role of ERRα as a regulator of mitochondrial function and OXPHOS is validated in neonatal cardiomyocytes, where a significant number of genes induced by ERRα expression are involved in cellular energy metabolic pathways [22]. In addition, a number of genes involved in mitochondrial fatty acid oxidation and lipid uptake are also induced by ERRα overexpression. Notably, medium chain acyl-CoA decarboxylase (MCAD), the rate limiting enzyme involved in fatty acid β-oxidation was confirmed as a direct transcriptional target for ERR by several other studies as well [10, 22].

Despite the differences in LBD, gene-chip analysis shows that ERRα and ERRγ target a common set of promoters of genes related to OXPHOS and fatty acid oxidation [31, 32]. In this context, both ERRs serve as positive transcriptional regulators of genes regulating mitochondrial respiration and fatty acid oxidation as well regulators for genes involved in gluconeogenesis [31, 33, 34]. In addition, ERRγ also positively regulate the promoters of G6Pase and PEPCK, two rate limiting enzymes of gluconeogenesis, while ERRα has been shown to be the transcriptional repressor of PEPCK [34]. ERRγ, the newest member identified in the ERR superfamily, plays a role in controlling metabolic switching in the perinatal heart and acts as a direct transcriptional regulator of GATA4 [35]. Compared to ERRα, which is associated with poor breast cancer outcomes, the overexpression of ERRγ was reported to associate with a better prognosis [36]. ERRγ was also identified as a potential tumor suppressor in gastric cancer by negatively regulating the Wnt signaling pathway [37]. Similar to ERRα, ERRγ plays a role in the regulation of mitochondrial gene expression [38].

In recent years, a role of ERRs in pluripotency has been identified primarily through studies of reprogramming of somatic cells to immortalized pluripotent stem cells (iPS) [39–41]. This was first recognized with ERRβ, which was found to replace Nanog or Klf4 during reprograming of iPS [39, 42]. In particular, ERRβ was found to bind to many target sites co-occupied by OCT4-SOX2-Nanog (OSN) [40, 41, 43], the transcriptional network characterized for their functions in maintaining "stemness". ERRβ participates in the regulation of these factors and their targets and is also a direct transcriptional target of Nanog [39]. Conversely, ERRβ also interacts with Oct4 within the *Nanog* promoter, a component also regulated by the Wnt/Gsk3 pathway [27, 41]. Further studies show that ERRβ is regulated by leukemia inhibitory factor (LIF), Wnt, PROX1, Ncoa3 as well as nucleostemin [44–48], all involved in pluripotency regulation.

*In vivo*, ERRβ is highly expressed during embryogenesis and is involved in the development and physiologic function of different tissues and organs, including the placenta, inner ear, and retina [49]. Embryos carrying homozygous deletions of ERRβ displayed impaired placental formation and died *in utero*, indicating that ERRβ plays a crucial role during early placental stages [50]. Consistently, knockdown or knockout of ERR allows for differentiation and calcium deposition while suppressing expression of genes associates with progenitor cells [51–53].

It was later reported that all three ERR isoforms are capable of supporting iPS reprogramming [54]. Using ERRα and γ and their cofactors to induce OXPHOS, it was demonstrated that at least an initial burst of the OXPHOS activity is necessary for the reprogramming of iPS [54]. ERRβ also regulates OXPHOS, similar to ERRα and γ [55]. Together, this work established that ERR transcriptional factors and the transcriptional network regulated by ERRs play an important role in the regulation of cell fate.
