Transcriptional regulation of gene expression
Gene expression in eukaryotic cells is regulated at transcriptional, post transcriptional and translational levels (Tomizawa, Lekstrom-Himes, and Xanthopoulos 17). Transcriptional control is the primary means of regulating gene expression. Eukaryotic transcriptional control functions at three levels: modulation of the activities of activators and repressors; variations in chromatin structure; and effect of activators and repressors on assembly of initiation complexes (Lodish et al. 368).
Regulation of transcription initiation is the most widespread form of gene control in eukaryotes. However, in some cases, transcription may be attenuated and regulated at subsequent steps, such as during or after the transcription process (Cooper n. pag.). Eukaryotic genes are regulated by multiple transcription control elements, including promoter-proximal elements and enhancers. In addition, various elements in the transcription-control regions regulate transcription of eukaryotic protein-coding genes. These elements include the TATA box, and initiator (Lodish et al.360).
Transcription is controlled by trans-acting proteins, called transcription factors, which bind at cis-acting regulatory DNA sequences. Transcription factors are equivalent to the repressors and activators in bacteria, which control the transcription of operons. Cis-acting DNA control elements are often located tens of thousands base pairs farther, either upstream or downstream, from the promoter they regulate. In that way, a transcription from a single promoter may be regulated by binding of multiple of transcription factors to alternative control elements, inducing the complex control of gene expression (Lodish et al. 360). The transcription factor also binds to the specific regulatory sequences and modulates the activity of RNA polymerase, which is an important enzyme during the transcription process. Another aspect in control of eukaryotic gene expression is the packaging of DNA into chromatin and its modification by methylation, so chromatin structure plays an important role in the process of gene expression regulation (Cooper n. pag.).
Particularly, in mammalian livers, some genes can response reversibly to external stimuli such as noxious chemicals. Genes are reversibly induced and repressed by transcriptional control in order to adjust the cell’s enzymatic machinery to its immediate nutritional and physical environment, which means genes are controlled in response to environmental variables, as occurs in bacteria. In general, in multicellular organisms, only a small fraction of genes respond to environmental changes if they are compared with single organisms, such as bacteria (Lodish et al. 359).
The regulation of specific gene expression in the liver is an active process and includes transcriptional control, post-transcriptional regulation and cell-cell contact (Panduro, Shalaby, and Shafritz 1172). Cell type-specific gene expression is controlled primarily at the level of transcription and involves several factors, such as CCAAT/enhancer binding proteins (C/EBPs) and some hepatocyte nuclear factor (HNFs). For example, the gene encoding HNF1α is controlled by HNF-4α (White 170). Promoters and enhancer regions are composed of multiple cis-acting DNA sequences, binding different HNFs to regulatory region of the gene and providing a synergy of transcriptional activation. These interactions play an important role in tissue-specific gene expression maintenance by the manifestation of distinct hepatocyte-specific target genes. Different amino acid sequences make up a structural motif in the DNA-binding domain of a transcription factor mediating the specific recognition of the DNA sequence. This specificity allows the recognition of DNA sites by the transcription factor, either in a proximal promoter or in distal enhancer sequences of hepatocyte-specific genes, defining the hepatocyte-specific genes regulated by a particular transcription factor (Costa et al. 1331).
Maintenance of liver-specific gene transcription is related to the cooperation of liver-enriched factors with the ubiquitous transactivating factors (Schrem, Klempnauer, and Borlak 140). In general, the stimulation of hepatocyte-specific gene transcription is associated to the binding of the liver-enriched transcription factors (C/EBP, HNF1, HNF3, HNF4, and HNF6) to multiple promote/enhancer sites, interacting synergistically one to each other (Costa et al. 1341)
Liver transcription factors (e.g.HNF4, HNF6)
Transcription factors are trans-acting DNA binding proteins that enable selective gene expression and regulation. These factors bind to a specific cis-acting DNA sequence in a regulatory element of a gene, interacting with the transcriptional machinery (Schrem, Klempnauer, and Borlak 130). Six major families of liver transcription factors families have been described, the “forkhead” protein gene family that HNF3 proteins (α, β, and γ) belong to: the CCAAT/enhancer binding proteins (C/EBP) is the original leucine zipper protein and includes other members, such as C/EBP/β and DBP, which are important in hepatocyte gene regulation; the HFN1 family includes HNF1α and HNF1β members, which are distantly related to the homeobox protein; the nuclear steroid-thyroid receptor superfamily, including the HFN4, which contains a zinc finger DNA binding domain; and the HFN-6. All of these liver transcription factors are important in the regulation of the expression of the genes, during the transcription process (Nagy, Bisgaard, and Thorgeirsson 223; Schrem, Klempnauer, and Borlak 140).
The liver-enriched transcription factors are grouped into related protein families according to the homology within DNA-binding domains. Numerous studies have established the important role of liver-enriched factors in the development of the organ and cellular function in liver-specific gene expression, all of which act at the same time and in tune with one another (Schrem, Klempnauer, and Borlak 294). In fact, they play important roles in liver developmental and survival (Costa et al. 1335)
The Forkhead box a1, Foxa 1, Foxa 2, and Foxa 3 proteins (previously named HNF3α, HNF3β, and HNF3 γ proteins) bind as a monomer to a DNA through the winged helix DNA-binding domain. They contain sequences for transcriptional activation and share more than 90% homology in their aminoacid sequence, binding to similar DNA sequences within specifics regulatory regions of the hepatocyte (Costa et al.1333).
Another transcription liver-enriched transcription factor is the HNF6 or ONECUT-1 (OC-1). This transcription factor contains a divergent homeodomain motif and a single cut domain. It binds as a monomer through the cut-homeodomain or the ONECUT DNA-binding domain to its DNA recognition site. It contains sequences for transcriptional activation and nuclear localization. Another ONECUT family member is the OC-2 that is expressed in the liver. OC-2 and OC1 share amino acid sequence homology and DNA-binding site specificity, providing functional redundancy in hepatocytes (Costa et al. 1333).
C/EBP are CCATT enhancer binding proteins, which utilize the amino-terminal basic region leucine zipper (bZIP), and a DNA binding interface containing basic amino acids. It is a heat stable DNA-protein found in rat liver nuclei, selectively binds to the CCAAT motif of some viral promoters (Schrem, Klempnauer, and Borlak, 295). Proteins, such as C/EBP α and C/EBP β are coexpressed in hepatocytes and have the ability to form either hetero or homodimes through the bZIP protein motif. Costa et al. 1332). The DBP belongs to the PAR family of bZip transcription factors (Bulla et al. 180)
The HNF1 α binds its DNA recognition sequence as a dimer by a POU-homeodomain sequence and a myosin-like dimerization domain. It also contains short acidic and basic sequences (Schrem, Klempnauer,and Borlak 139). In the liver, HNF1 α is coexpressed with HNF1 β, forming heterodimes with the HNF1 β-related family member.
The liver-enriched transcription factor hepatocyte nuclear factor-4 (HFN-4) utilizes the zinc finger DNA-binding domain to recognize DNA. Zinc-fingers are small DNA-binding peptide motifs and can be used to construct larger protein domains that bind to specific DNA sequences (Schrem, Klempnauer, Borlak 139).
Other liver-enriched transcription factors, such as the Nkx-2.8 homeodomain family member, α -fetoprotein promoter- linked coupling element factor, and the fetoprotein transcription factor (FTF), have been identified through DNA analysis (Costa et al. 1333).
Locus control regions
Locus control regions (LCR) are cis-regulatory elements with the ability to enhance the expression of genes that are linked at distal chromatin sites (Li et al. 3077). LCR and gene-proximal elements can modified the expression level of genes, including promoters, enhancers and silencers. They are complex transcriptional regulators, which control the gene clusters in mammalian genes including serpin clusters (Zhao, Friedman, and Fournier 5286). In rats, LCR such as the aldolase C gene, whey acidic protein (WAP) gene, kallikrein genes, and LAP(C/EBP β) gene have been identified (Li et al. 3086).
The Serpins (serine protease inhibitor) are a superfamily of gene sequence encoding protein products that achieve different functions in vivo and have a different forms of regulation depending on the cell type (Marsden and Fournier, 1768). In rats, three serine-protease inhibitor gene families, SPI 2.1, SPI 2.2 and SPI 2.3, are expressed in liver in a specific, hormonally controlled manner; they are also called SP1, SP2, and SP3. Another hepatic serpin gene is the α1 antitrypsin (Schwarzenberg et al. C1144). Physiological status of the animal influences this expression (Paquereau et al. 1053). There are 4 or 5 genes at each of Spi loci (Festoff 139). Proximal promoter sequences on the 5’fkanking SPI gene sequences are strongly conserved in all three SPI genes (Rossi et al. 1061). Starvation influence in the decrease of mRNA was only found in SP1 2.1, while for Spi 2.2, 2.3 and α 1 antitrypsin, mRNA did not change. Their expression did not depend on androgens, as occurs in mouse contrapsin, which is in close homology to the Spi genes (Schwarzenberg et al. C1144).
In normal rats, the SPI 2.1 and SPI 2.2 gene arcs express at maximal level, and is repressed in hypophysectomized and highly inflamed rats. This is contrary to the SPI 2.3 gene, which is almost silent in control rats and induced in treated rats (Paquereau et al. 1053). Expression of SPI gene is mainly controlled by several hormones, such as growth hormone and glucocorticoid, which act as potentiating factors and regulate the transcription of the SPI 2.1 and SPI 2.2 genes (Rossi et al. 1061).
In the SPI 2.3 gene, an additional 42 bp element is present. This gene escapes growth hormone regulation. The regulation of this gene’s expression is complex, involving positive promoter-regulatory elements and negative regulatory ones. The positive regulatory elements include some interkeukin-6-response sites and glucocorticoid (CG)-response element. On the other hand, the negative regulatory is located in the 39 untranslated region (Simar-Blanchet et al. 392).
Cell culture system
Two types of epithelial cells are present in the liver: hepatocytes, 60-70%, and intrahepatic bile duct epithelial cells or cholangiocytes (2 to 3%). Cells can be cultured outside from their natural environment. During that process, controlled conditions allow cell growth. Different technologies have been applied for cell culture including 3D-cell culture, which is closer to in vivo natural system (Ravi et al. 16)
Liver function, disease, pharmacology and other related subjects can be done using primary hepatocyte culture. These cells can be recovered from adult rats by hepatocytes dissociation and then filtered through a 100 μm pore size mesh nylon filter. Cells are cultured onto plates using some medium (Sheng et al. 3917).
Although the regenerative capacity of rat liver in adult rat is high, isolated cell proliferation is limited in vitro. A subpopulation of hepatocyte, named small hepatocytes has a higher growth potential limited to a period of 1 or 2 weeks after isolation. Another possibility of liver culture is the cell lines derived from rat liver stem cell (LSC) which circumvent the problem of the low time survival and reduce the usage of experimental animal (Kujik, n. pag.)
Microarray for gene expression
Microarray allows for the measurement of mRNA levels in a cell of tissue using many genes at the same time. A typical microarray experiment involves the hybridization of an RNA molecule to the DNA template from which it is originated. Many samples of DNA are used to construct an array. Expression level of the genes is determined by the amount of DNA joined in each site. Results are collected and a profile for the expression of the gene in the cell is generated. In that way, a simultaneous analysis of mRNA expression level of many genes is generated by this technique (Premier Biosoft n. pag.).
Two different formats of microarray-based technologies are available, depending on the target nucleic acid component, the oligonucleotide array and the cDNA microarray. The oligonucleotide array is based on the generation or oligonucleotide targets, in situ, on a solid surface attached to the glass surface by photochemical removing protecting groups. In the cDNA microarray, a single strand of cDNA for the gene of interest is immobilized on spots organized in a grid, array, or a support, such as glass, quartz wafer or nylon membrane. mRNA is extracted from the sample of interest, labeled and hybridized to the array. Label intensity in each spot is quantified and the amount of mRNA produced is determined. Labeling can be done by using either radioactive or fluorescence labels, or by classifying two samples using green and red fluorescent dye, Standford technology (Huber, Von Heydebreck, and Vingron n. pag.). Although both techniques are useful, cDNA microarray is used for the screening of mRNA expression levels and oligonucleotide array can be used for a more precise analysis such as the detection of a single nucleotide polymorphism (Lida and Nishimura 35).
Different types of microarray are used, including microarray expression analysis, microarray for mutation analysis, and comparative genomic hybridization. In the microarray expression analysis, the cDNA from the mRNA of known genes, normal and the diseased genes, are immobilized. When the disease occurs, an overexpression of the gene is visualized by spots with more intensity in the disease tissue gene (Premier Biosoft n.p.).
The critical aspects of the microarray technique are the fabrication of a cDNA microarray, and the probe preparation and microarray hybridization. In the fabrication of cDNA microarray, the cDNA is prepared as arrayed targets, using either single or double stranded cDNA. The length of this DNA is from 0.2 to 2. 5 Kb, and the concentration should be as high as 500 ng/µL. cDNA is attached to the coated surface and the arraying technique can be by passive dispensing or drop-on demand delivery. For the probe preparation and microarray hybridization, the total mRNA or mRNA is isolated from the cells or the tissue, as 15 to 200 µg of RNA are necessary. RNA is converted in cDNA by reverse transcription and labeled with the dye and hybridized (Lida and Nishimura 35).
Investigators have turned to this methodology to investigate phenomena particularly appropriate for the analysis of expressed liver genes. Jee et al. observed, using microarray analysis, that some genes respond to a sustained reduction of the insulin levels in the liver, concluding that microarray testing is a useful tool to better understand the insulin-regulated diabetes-related diseases (829). Also, microarray studies have been a helpful technique for obtaining knowledge of molecular mechanisms related to hepatocellular carcinoma by the identification of novel molecular subgroups (Maass et al. 261).
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