**2. Transcriptional regulation of urea cycle genes**

## **2.1 Transcriptional regulation of mammalian NAGS gene**

Although the existence of mammalian NAGS gene and its product have been known since the 1950s [44], the gene remained elusive until 2002, when it was identified and cloned in mice and humans [45–48]. The human *NAGS* gene is located on chromosome 17 within band 17q21.31 and spans approximately 8.5 kb. This includes seven exons that encode a 1605 bp open reading frame, six introns, a promoter, and an enhancer located about 3 kb upstream of the transcription start sites [45, 47, 49–51]. The mouse *Nags* gene is located in the syntenic region on chromosome 11. Pairwise BLAST [52] was used for comparison of the regions upstream of the NAGS genes from seven mammals including human; this analysis revealed two conserved elements, one located immediately upstream of the first exon of

**85**

*Data Mining Approaches for Understanding of Regulation of Expression of the Urea Cycle Genes*

the *NAGS* gene and a putative regulatory element located about 3 kb upstream of the *NAGS* translation initiation site [50]. The pattern of DNA sequence conservation within the conserved region immediately upstream of the first exon of the *NAGS* gene suggested that it might consist of a promoter and a proximal regulatory element, which is similar to the CPS1 regulatory region that will be described in the next section. Cis-element over-representation (CLOVER) software [53] was then used to identify binding sites for specificity protein 1 (Sp1), cAMP response element binding/activating transcription factor (CREB/ATF), and CCAAT-enhancer binding protein (C/EBP) transcription factors in the putative *NAGS* promoter, while activator protein-2 (AP2), hepatic nuclear factor 1 (HNF1), nuclear factor-Y (NF-Y), and mothers against decapentaplegic homolog 3 (SMAD3) binding sites were found in the upstream regulatory element, which was named −3 kb enhancer.

Reporter gene assays were used to confirm that conserved regions located adjacent to and −3 kb upstream of the first *NAGS* exon indeed function as a promoter and enhancer in the HepG2 hepatoma cells [50]. Consistent with the absence of the TATA-box in the NAGS promoter, transcription of the *NAGS* mRNA in the liver and intestine initiates at multiple sites located between 50 and 150 bp upstream of the NAGS translation initiation codon [50]. Binding of the Sp1 and CREB transcription factors to the *NAGS* promoter, and binding of the HNF1 and NF-Y transcription factors to the −3 kb enhancer were confirmed with chromatin immunoprecipitation (ChIP) and DNA pull-down assays [50]. Binding of HNF1 to the −3 kb NAGS enhancer is responsible for the liver-specific expression of the *NAGS* gene [50] and the role of HNF1 transcription factor in expression of the *NAGS* gene was confirmed when a sequence variant that caused decreased HNF1 binding to its site was

Regulatory region of the rat *Cps1* gene has been cloned in 1985 [54]. Almost all of our knowledge of transcriptional regulation of the CPS1 gene is based on experiments with the rat *Cps1* gene in the rat and human hepatoma cell lines and transgenic mice. The aim of these studies was to elucidate the mechanism of regulation of *Cps1* expression by glucagon and glucocorticoids as well as identify regulatory

A promoter, located immediately upstream of the first *Cps1* exon, a proximal enhancer that is immediately adjacent to the promoter, a distal enhancer located about 6 kb upstream of the *Cps1* translation initiation codon, and another regulatory element located about 10 kb upstream of the *Cps1* translation initiation site are responsible for transcriptional regulation of the rat *Cps1* gene [27, 29, 56, 58]. Transcription of the rat *Cps1* mRNA is initiated 138–140 bp upstream of the translation initiation site by the promoter that has TATA and CAAT motifs [59] and binds C/EBP transcription factor [29]. The distal *Cps1* enhancer consists of a cAMP response unit (CRU) and a glucocorticoid response unit (GRU); each response unit binds multiple transcription factors that activate *Cps1* expression in response to glucagon and glucocorticoids, and are responsible for *Cps1* expression in periportal hepatocytes [26, 55, 56, 60]. The CRU binds CREB, HNF3, C/EBP transcription factors, and a yet to be identified protein P1 [60, 61], while GRU binds glucocorticoid receptor, hepatocyte nuclear factor 3/forkhead box A (HNF3/FOXA), C/EBP, a 75 kDa protein P3, and a yet to be identified protein P2 [26, 61, 62]. The distal *Cps1* enhancer activates *Cps1* transcription via proximal enhancer that binds C/EBP and glucocorticoid receptor and is located immediately upstream of the *Cps1* promoter [63]. These studies of the rat *Cps1* gene regulation rest on the premise that the rat

elements that restrict *Cps1* expression to periportal hepatocytes [55–57].

*DOI: http://dx.doi.org/10.5772/intechopen.81253*

These findings were then experimentally verified.

found in a patient with NAGS deficiency [49].

**2.2 Transcriptional regulation of mammalian CPS1 gene**

#### *Data Mining Approaches for Understanding of Regulation of Expression of the Urea Cycle Genes DOI: http://dx.doi.org/10.5772/intechopen.81253*

the *NAGS* gene and a putative regulatory element located about 3 kb upstream of the *NAGS* translation initiation site [50]. The pattern of DNA sequence conservation within the conserved region immediately upstream of the first exon of the *NAGS* gene suggested that it might consist of a promoter and a proximal regulatory element, which is similar to the CPS1 regulatory region that will be described in the next section. Cis-element over-representation (CLOVER) software [53] was then used to identify binding sites for specificity protein 1 (Sp1), cAMP response element binding/activating transcription factor (CREB/ATF), and CCAAT-enhancer binding protein (C/EBP) transcription factors in the putative *NAGS* promoter, while activator protein-2 (AP2), hepatic nuclear factor 1 (HNF1), nuclear factor-Y (NF-Y), and mothers against decapentaplegic homolog 3 (SMAD3) binding sites were found in the upstream regulatory element, which was named −3 kb enhancer. These findings were then experimentally verified.

Reporter gene assays were used to confirm that conserved regions located adjacent to and −3 kb upstream of the first *NAGS* exon indeed function as a promoter and enhancer in the HepG2 hepatoma cells [50]. Consistent with the absence of the TATA-box in the NAGS promoter, transcription of the *NAGS* mRNA in the liver and intestine initiates at multiple sites located between 50 and 150 bp upstream of the NAGS translation initiation codon [50]. Binding of the Sp1 and CREB transcription factors to the *NAGS* promoter, and binding of the HNF1 and NF-Y transcription factors to the −3 kb enhancer were confirmed with chromatin immunoprecipitation (ChIP) and DNA pull-down assays [50]. Binding of HNF1 to the −3 kb NAGS enhancer is responsible for the liver-specific expression of the *NAGS* gene [50] and the role of HNF1 transcription factor in expression of the *NAGS* gene was confirmed when a sequence variant that caused decreased HNF1 binding to its site was found in a patient with NAGS deficiency [49].

## **2.2 Transcriptional regulation of mammalian CPS1 gene**

Regulatory region of the rat *Cps1* gene has been cloned in 1985 [54]. Almost all of our knowledge of transcriptional regulation of the CPS1 gene is based on experiments with the rat *Cps1* gene in the rat and human hepatoma cell lines and transgenic mice. The aim of these studies was to elucidate the mechanism of regulation of *Cps1* expression by glucagon and glucocorticoids as well as identify regulatory elements that restrict *Cps1* expression to periportal hepatocytes [55–57].

A promoter, located immediately upstream of the first *Cps1* exon, a proximal enhancer that is immediately adjacent to the promoter, a distal enhancer located about 6 kb upstream of the *Cps1* translation initiation codon, and another regulatory element located about 10 kb upstream of the *Cps1* translation initiation site are responsible for transcriptional regulation of the rat *Cps1* gene [27, 29, 56, 58]. Transcription of the rat *Cps1* mRNA is initiated 138–140 bp upstream of the translation initiation site by the promoter that has TATA and CAAT motifs [59] and binds C/EBP transcription factor [29]. The distal *Cps1* enhancer consists of a cAMP response unit (CRU) and a glucocorticoid response unit (GRU); each response unit binds multiple transcription factors that activate *Cps1* expression in response to glucagon and glucocorticoids, and are responsible for *Cps1* expression in periportal hepatocytes [26, 55, 56, 60]. The CRU binds CREB, HNF3, C/EBP transcription factors, and a yet to be identified protein P1 [60, 61], while GRU binds glucocorticoid receptor, hepatocyte nuclear factor 3/forkhead box A (HNF3/FOXA), C/EBP, a 75 kDa protein P3, and a yet to be identified protein P2 [26, 61, 62]. The distal *Cps1* enhancer activates *Cps1* transcription via proximal enhancer that binds C/EBP and glucocorticoid receptor and is located immediately upstream of the *Cps1* promoter [63]. These studies of the rat *Cps1* gene regulation rest on the premise that the rat

*Gene Expression and Control*

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**Figure 2.**

**2. Transcriptional regulation of urea cycle genes**

**2.1 Transcriptional regulation of mammalian NAGS gene**

*transcriptional factor; CREB—cAMP response element binding transcription factor.*

Although the existence of mammalian NAGS gene and its product have been known since the 1950s [44], the gene remained elusive until 2002, when it was identified and cloned in mice and humans [45–48]. The human *NAGS* gene is located on chromosome 17 within band 17q21.31 and spans approximately 8.5 kb. This includes seven exons that encode a 1605 bp open reading frame, six introns, a promoter, and an enhancer located about 3 kb upstream of the transcription start sites [45, 47, 49–51]. The mouse *Nags* gene is located in the syntenic region on chromosome 11. Pairwise BLAST [52] was used for comparison of the regions upstream of the NAGS genes from seven mammals including human; this analysis revealed two conserved elements, one located immediately upstream of the first exon of

*Transcriptional regulatory elements of the urea cycle genes. Hooked arrows indicate transcriptional initiation sites. Gray boxes—cisacting regulatory elements. Proteins that bind to cis-acting regulatory elements are shown above each box. C/EBP—CCAAT/enhancer binding proteins; HNF4α—hepatic nuclear factor a; COUP-TF chicken ovalbumin upstream promoter transcriptional factor; FoxA—forkhead box A transcriptional factor/ hepatic nuclear factor 3; GR—glucocorticoid receptor; P3—unidentified protein of approx. 75 kDa; P1, P2—two unidentified proteins; NF-Y—CCAAT—binding factor; AP-2—activator protein 2; Sp1—Sp1* 

gene is a good model for human *CPS1*. However, the two species have different metabolic rates due to their different sizes and regulation of the CPS1 gene and urea cycle may differ in the two organisms. More recently, a region of the human *CPS1* gene that corresponds to the rat *Cps1* promoter and proximal enhancer has been shown to bind HNF3 and direct reporter gene expression in hepatoma cells [64]. Human *CPS1* gene is located on chromosome 2, band 2q34 where it spans approx. 125 kb and has 38 exons that encode a 1500 amino acids long protein.
