Eukaryopedia

As most synthetic biologists and iGEM teams work with Escherichia Coli, the use of other model systems can create confusion. We hope to ease the legibility of our project descriptions by creating eukaryopedia, an overview about transcription factors and cell lines we used in our studies. We hope it can help you find guidance in the jungle that mammalian molecular biology is at the moment.

Cell lines

MCF-7

MCF-7 is a hormone-dependent, poorly invasive human breast cancer cell line [1]. Originally, the cell line was derived from a postmenopausal woman with metastatic breast cancer at the Michigan Cancer Foundation. It was observed, however, that cell lines used in different laboratories vary greatly in their biological characteristics, so that it is suggested that they were derived from different patients [2]. MCF-7 cells are estrogen-receptor positive and require estrogen for tumorigenesis in vivo. 17β-estradiol induces an TGFα-like activity [3], which promotes tumor growth and progression [4]. Furthermore the cells express receptors for and respond to several other hormones including androgen, progesterone, glucocorticoids, insulin, epidermal growth factor, insulin-like growth factor, prolactin and thyroid hormone [2].

HeLa

The cells were originally derived in 1952 from Henrietta Lacks, who suffered from an adenocarcinoma of the cervix. The HeLa cells were the first human epithelial cells established in long-term culture [5]. There are three main characteristics of the genome of HeLa by which they can be recognized: hypertriploid chromosome number (3n+), 20 clonally abnormal chromosomes and the integration of multiple copies of HPV18 (Human Papilloma Virus) at various sites [6]. It has been shown, that the HeLa genome has been remarkably stable after years of subcultivation [6], but it is also possible to select strains of HeLa cells with certain properties by putting them under selection pressure [7].

U2-OS

U2-OS, formerly known as 2T cell line [8], were derived from a 15-year-old girl with a moderately differentiated osteogenic sarcoma of the shinbone. Cell culture of U2-OS started at the time of amputation of the left leg on September 3, 1964 [9]. U2-OS cells express adhesion molecules such as integrins, Ig-CAMs and chemokine receptors as well as growth factors which are either constitutively expressed (such as IL-7) or inducible (such as TNF) by PMA (phorbol ester) or ionomycine. The adhesion molecules and growth factors support the growth of CD34 progenitor cells [10].

Transcription factors

NF-κB

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a transcription factor (TF) which regulates many different target genes resulting in the expression of various proteins. In most cell types (with the exception of B cells and Dentritic cells) NF-κB is bound to the Inhibitor of κB (IκB), which withhold NF-κB from entering the nucleus. When the cell becomes activated by an extra cellular stimluli, IκB is degraded and NF-κB can enter the nucleus. Within the nucleus NF-κB is able to enhance transcription of genes which are involved in immune response, cell proliferation or cell survival, depending on cell type and extra cellular stimuli [11]. In many cells NF-κB regulates anti-apoptotic proteins (e.g. TRAF1/2) and therby preventing cell death. Therefore mutations of NF-κB resulting in a constitutively active form are often associated with unregulated cell proliferation and cancer [12]. In macrophages the NF-κB signalling pathway could be activated by binding of bacterial lipopolysacchride (LPS). There NF-κB activation leads to secretion of cytokines which influence other lymphocytes.

p53

P53 is a transcription factor (TF) which is involved in several physiological processes. One major function of P53 is cell cycle regulation. P53 is often activated through DNA damage or other cellular stresses like cell cycle abnormalities, hypoxia and oxidative stress. In normal cells the P53 level is kept low by the protein HDM2, which attaches ubiqutin to P53 (acts as ubiquitin ligase). The ubiquitylation of P53 leads to its degradation by the proteasome. In response to cellular stress P53 is phosphorylated and changes its conformation in way preventing HDM2 binding. Conformational changes also result in the exposion of the DNA binding domain. This activation of P53 leads to farreaching alteration of gene expression. The cell cycle is stopped between G1 and S phase and DNA repair systems are switched on. If the cell damage is intense P53 accumaltion can also lead to apoptosis of the cell. Because of its roles in DNA protection and cell cycle regulation P53 mutation is often correlated with cancer [13].

HIF-1

HIF-1 (hypoxia inducible factor-1) is a transcription factor (TF) which is exclusivly active during hypoxia (low oxygen level). HIF-1 is a heterodimer consisting of a α- and a β-subunit. During normal oxygen conditions the α-subunit is hydroxylated by the HIF prolyl-hydroxylase. The hydroxylated α-subunit is a target for an ubiquitin ligase. Ubiquitylation of the α-subunit leads to its degradation by the proteasome. During hypoxia the degradation of the α-subunit does not occur, since the HIF prolyl-hydroxylase uses oxygen as a cosubstrate. The active TF enhances expression of different genes as for instance genes associated with blood vessel formation.

pPARγ

Peroxisome proliferator-activated receptor γ (PPAR γ) is a transcription factor belonging to the family of nuclear receptors. PPAR γ plays an important role in glucose metabolism and fatty acid storage. PPAR γ is basically activated by ligands like the prostaglandin PGJ2 and through dimerization with retinoid X receptor (RXR)[14]. The activated heterodimer binds to the DNA consensus sequence AGGTCANAGGTCA resulting in an increased or decreased transcription of the appropriate gene. The genes activated by PPAR γ initiate the uptake of fatty acids and differentiation of cells to adipocytes. Besides its function in metabolism, PPAR γ was also shown to be correlated with several diseases such as cancer and diabetes. Activation of PPAR γ by synthetic PPAR γ ligands result in an increased glucose uptake. These syntethtic ligands are therefore promising agents in diabetes II treatment [15]. Another synthetic ligand of PPAR γ is able to inhibit the proliferation of different cancer types [16].

SREBP

Sterol regulatory element-binding protein (SREBP) is a transcription factor involved in the regulation of sterol metabolism. In cells with high concentration of cholestrol SREBP is present in an inactive form anchored to the endoplasmatic reticulum or the nuclear envelop. If the cholesterol concentration decreases SREBP is cleaved by the proteases site-1 protease and site-2 protease resulting in a release of the aminoterminal domain of SREBP. Two additional proteins (Scap and Insig) are needed to regulate this process in a way that the cleavage occurs exclusively during lack of sterol[17]. The aminoterminal domain of SREBP is translocated into the nucleus and binds to the DNA consensus sequence TCACNCCAC. The binding causes an up regulation of the genes needed for cholesterol synthesis.

Sp1

Specificity protein 1 (Sp1) is a transcription factor which belongs to the zinc-finger protein family. It binds to promoter elements containing a central CpG motive with the following consensus sequence; 5'-G/TGGGCGGG/AG/AC/T-3' [18]. Sp1 is involved in chromatin-remodelling processes [19] as well as in derecruiting repressor proteins from the promoter [20]. For these and for other reasons Sp1 is often considered as a universal transcription supporting protein. Sp1 was shown to regulates various genes responsible for cellular processes like apoptosis, cell growth and differentiation and immune response [21]. It interacts with several well known proteins, such as c-myc, c-Jun and Stat1 [21].

Ap1

Activator Protein1 (Ap1) is a transcription factor which is activated by several pathways and extracellular stimuli like UV radiation, growth factors and bacterial and viral infections. Ap1 is a heterodimer consisting of one member of the Fos and one member of the Jun family. Ap1 composition and activation is mainly controlled by MAP kinase cascades by up regulating the expression of both Fos and Jun proteins. Besides the heterodimerisation process, phosphorylation of the complex is needed to achieve an efficient transcription of the target genes [22]. Activated Ap1 binds to DNA sequences with the consensus sequence 5'-TGAG/CTCA-3' [23]. The genes regulated by Ap1 are involved in cellular processes like apoptosis, cell differentiation, cell proliferation and oncogenic transformation[22].

CREB

CAMP responsive element binding protein (CREB) is a transcription factor which binds to cAMP response elements (consensus sequence 5'- TGACGTCA -3' [24] occurring in many promoter sequences. CREB is activated by MAP kinase cascade but also through the cAMP signalling pathway. Homodimerisation leads finally to an activated complex and binding to DNA occurs via a leucine zipper domain (s. Picture). Many genes are regulated by CREB including the neurotrophin Brain-derived neurotrophic factor, c-fos and some neuropeptides. CREB is thought to be involved in processes like long-term memory [25] and drug addiction [26]. CREB plays also an important role in cell survival [27].

NF-Y

Nuclear factor Y (NF-Y), also called core binding factor (CBF) is a transcription factor which binds to the consensus sequence CCAAT [29] occurring in about 25% of eukaryotic genes [28]. NF-Y is involved in the transcription regulation of several genes, including HSP70, albumin, FGF-4, α-collagen, β-actin and several others [29]. NF-Y is a heterotrimeric complex and is evolutionary extremely conserved. It was also shown that NF-Y plays a major role in cellular senescence [28].

RNA-processing and transcriptional regulation

Post-transcriptional modification / mRNA processing in eukaryotes

To express a gene and successfully synthesize the appropriate protein the gene must firstly transcript into mRNA. Unlike in bacteria, this mRNA molecule is not directly ready for translation; the primary transcript is therefore called precursor-mRNA (pre-mRNA). One of the first modifications is a process referred to as 5’-capping. By means of several biochemical steps a 7-methylguanosine molecule is bound to the 5’ end of the pre-mRNA, via a 5’ to 5’ triphoshpate linkage. This 5’ cap has various functions including prevention of 5’ degradation, export from the nucleus and initiation of translation. Not only the 5’ end but also the 3’ end is modified, this process is called polyadenylation. Therefore a Polyadenylation signal is needed (consensus sequence 5'- AAUAAA-3'), further in the 3’ direction occurs a 5’-CA-3’ element, these both sequences are recognized by the enzymes cleavage and polyadenylation specificity factor and cleavage stimulation factor. Together they are attracting many other proteins including Polyadenylate Polymerase (PAP). The protein complex cuts the pre-mRNA at the CA element and the PAP adds about 200 adenine residues to the 3’ end. The function of the poly-A tail is protection against degradation, marking of the end of the transcript and aid in translation initiation. The pre-mRNA contains not only these sequences coding for the protein, so called exons, but also many sequences which are non-coding. These introns have to be removed, that occurs in a process known as splicing. A protein complex called spliceosom connects all the exons thereby cutting out the introns. Responsible for the recognition of the exon-intron borders are small nuclear RNA within the spliceosom. Many genes can be spliced in several ways, an incident termed alternative splicing. [30,31]

Regulation of transcription in eukaryotic organisms

Cells have to adapt to changes in their environment and must be able to receive and react to extra cellular signals; cells accomplish these requirements by the up and down regulation of certain proteins. The protein expression in eukaryotic cells can be regulated on many different levels, this article concentrate on the regulation of transcriptions. Only a small percentage of the human genomic DNA is transcribed into mRNA. On the opposite, a huge part of the human genome is involved in regulating the transcription of coding sequences. To initiate transcription of a gene eukaryotic RNA-polymerases have to bind to several general transcription factors to establish the so called initiation complex (IC), which is able to bind to the DNA. The binding occurs upstream of the transcriptional start site (TSS) in a region called core promoter, this part of the promoter often contains specific elements like the TATA-Box (consensus sequence, TATAA/TAA/T, about 30 bp upstream of TSS [32]) and the GC-Box (consensus sequence TGTGGCTNNNAGCCAA) app. 80 bp upstream of the TSS [33] to which the IC can bind. Further upstream is a part of the promoter which is referred to as proximal promoter. Containing specific sequence elements, this part of the promoter is highly important for the transcriptional regulation. Transcription factors can bind to these response elements thereby up regulating or down regulating the gene transcription. Proteins methylating or acetylating the DNA are also involved in gene transcription regulation by remodelling of the Chromatin structure.

References

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[3] Dickson, R. B., Bates, S. E., McManaway, M. E. & Lippman, M. E. Characterization of Estrogen Responsive Transforming Activity in Human Breast Cancer Cell Lines. Cancer Research 46, 1707-1713 (1986).

[4] Booth, B. W. & Smith, G. H. Roles of transforming growth factor-α in mammary development and disease. Growth Factors 25, 227-235 (2007).

[5] Gey, G. O., Coffman, W. D. & Kubicek, M. T. Tissue culture studies of the proliferative capacity of cervical carcinoma and norml epithelium. Cancer Research 12, 264-265 (1952).

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[9] Ponten, J. & Saksela, E. Two established in vitro cell lines from human mesenchymal tumours. International Journal of Cancer 2, 434-447 (1967).

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[15] H. Phillip Koeffler. Peroxisome Proliferator-activated Receptor and Cancers. Clinical Cancer Research 9, 1-9 (2003).

[16] Suh, N. et al. A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9- dien-28-oic acid, with potent differentiating, antiproliferative, and antiinflammatory activity. Cancer Res. 59, 336–341 (1999).

[17] Brown MS, Goldstein JL . The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89 (3), 331–40 (1997).

[18] Briggs, M. R., J. T. Kadonaga, S. P. Bell, and R. Tjian. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science 234, 47-52. (1986).

[19] Stielow, B., A. Sapetschnig, C. Wink, I. Kruger, and G. Suske. SUMO-modified Sp3 represses transcription by provoking local heterochromatic gene silencing. EMBO Rep. 9, 899-906 (2008).

[20] Zhang, Y., M. Liao, and M. L. Dufau. Unlocking repression of the human luteinizing hormone receptor gene by trichostatin A-induced cell-specific phosphatase release. J. Biol. Chem. 283, 24039-24046 (2008).

[21] Nicole Y. Tan and Levon M. Khachigian. Sp1 Phosphorylation and Its Regulation of Gene Transcription. Molecular and Cellular Biology 29, 2483-2488 (2009).

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[28] Matuoka K, Chen KY. Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y. Ageing Res Rev. 1(4), 639-51 (2002).

[29] A Ronchi, M Bellorini, N Mongelli, and R Mantovani. CCAAT-box binding protein NF-Y (CBF, CP1) recognizes the minor groove and distorts DNA. Nucleic Acids Res. 25, 23(22), 4565–4572 (1995).

[30] Moore MJ, Proudfoot NJ.Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136(4), 688-700 (2009).

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[33] Litt M, Qiu Y, Huang S. Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation. 29(2), 131-41 (2009).