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An E-box (enhancer box) is a Response element found in some eukaryotes that acts as a protein-binding site and has been found to regulate gene expression in neurons, muscles, and other tissues. Its specific DNA sequence, CANNTG (where N can be any nucleotide), with a palindromic canonical sequence of CACGTG, is recognized and bound by transcription factors to initiate gene transcription. Once the transcription factors bind to the promoters through the E-box, other enzymes can bind to the promoter and facilitate transcription from DNA to mRNA.
In 1989, David Baltimore's lab discovered the first two E-box binding proteins, E12 and E47. These immunoglobulin enhancers could bind as Protein dimer to proteins through bHLH domains. In 1990, another E-protein, ITF-2A (later renamed E2-2Alt) was discovered that can bind to immunoglobulin light chain enhancers. Two years later, the third E-box binding protein, HEB, was discovered by screening a cDNA library from HeLa cells. A splice-variant of the E2-2 was discovered in 1997 and was found to inhibit the promoter of a muscle-specific gene.
Since then, researchers have established that the E-box affects gene transcription in several eukaryotes and found E-box binding factors that identify E-box consensus sequences.Mädge B.: E-Box. In: Schwab M. (Ed.) Encyclopedia of Cancer. Springer-Verlag Berlin Heidelberg, 2009. In particular, several experiments have shown that the E-box is an integral part of the transcription-translation feedback loop that comprises the circadian clock.
The E-box binding is modulated by Zn2+ in mice. The CT-Rich Regions (CTRR) located about 23 nucleotides upstream of the E-box is important in E-box binding, transactivation (increased rate of genetic expression), and transcription of circadian genes BMAL1/NPAS2 and BMAL1/CLOCK complexes.
The binding specificity of different E-boxes is found to be essential in their function. E-boxes with different functions have a different number and type of binding factor.
The consensus sequence of the E-box is usually CANNTG; however, there exist other E-boxes of similar sequences called noncanonical E-boxes. These include, but are not limited to:
The E-box plays an important role in circadian genes; so far, nine E/E'BOX controlled circadian genes have been identified: PER1, PER2, BHLHB2, BHLHB3, CRY1, DBP, Rev-ErbA alpha, Rev-ErbA beta, and RORC. As the E-box is connected to several circadian genes, it is possible that the genes and proteins associated with it are "crucial and vulnerable points in the (circadian) system."
The E-box is one of the top five transcription factor families associated with the circadian phase and is found in most tissues. A total of 320 E-box-controlled genes are found in the SCN (suprachiasmatic nucleus), liver, aorta, adrenal, WAT (white adipose tissue), brain, atria, ventricle, prefrontal cortex, skeletal muscle, BAT (brown adipose tissue), and calvarial bone.
E-box like CLOCK-related elements (EL-box; GGCACGAGGC) are also important in maintaining circadian rhythmicity in clock-controlled genes. Similarly to the E-box, the E-box like CLOCK related element can also induce transcription of BMAL1/CLOCK, which can then lead to expression in other EL-box containing genes (Ank, DBP, Nr1d1). However, there are differences between the EL-box and the regular E-box. Suppressing DEC1 and DEC2 has a stronger effect on E-box than on EL-box. Furthermore, HES1, which can bind to a different consensus sequence (CACNAG, known as the N-box), shows suppression effect in EL-box, but not in E-box.
Both non-canonical E-boxes and E-box-like sequences are crucial for circadian oscillation. Recent research on this forms an hypothesis that either a canonical or non-canonical E-box followed by an E-box like sequence with 6 base pair interval in between is a necessary combination for circadian transcription. In silico analysis also suggests that such an interval existed in other known clock-controlled genes.
Knowing that binding activates transcription of the per gene in the promoter region, researchers discovered in 2002 that DEC1 and DEC2 (bHLH transcription factors) repressed the CLOCK-BMAL1 complex through direct interaction with BMAL1 and/or competition for E-box elements. They concluded that DEC1 and DEC2 were Regulator gene of the mammalian molecular clock.
In 2006, Ripperger and Schibler discovered that the binding of this complex to the E-box drove circadian DBP transcription and chromatin transitions (a change from chromatin to facultative heterochromatin). It was concluded that CLOCK regulates DBP expression by binding to E-box motifs in enhancer regions located in the first and second introns.
In 1991, researchers tested whether c-Myc could bind to DNA by dimerizing it to E12. Dimers of E6, the Chimeric gene protein, were able to bind to an E-box element (GGCCACGTGACC) which was recognized by other HLH proteins. Expression of E6 suppressed the function of c-Myc, which showed a link between the two.
In 1996, it was found that Myc heterodimerizes with MAX and that this heterodimeric complex could bind to the CAC(G/A)TG E-box sequence and activate transcription.
In 1998, it was concluded that the function of c-Myc depends upon activating transcription of particular genes through E-box elements.
When MyoD binds to the E-box motif CANNTG, muscle differentiation and expression of muscle-specific proteins is initiated. The researchers ablated various parts of the recombinant MyoD sequence and concluded that MyoD used encompassing elements to bind the E-box and the tetraplex structure of the promoter sequence of the muscle specific gene α7 integrin and sarcomere sMtCK.
MyoD regulates HB-EGF (Heparin-binding EGF-like growth factor), a member of the EGF (Epidermal growth factor) family that stimulates cell growth and proliferation. It plays a role in the development of hepatocellular carcinoma, prostate cancer, breast cancer, esophageal cancer, and gastric cancer.
MyoD can also bind to noncanonical E boxes of MyoG and regulate its expression.
MyoG and MyoD have also been shown to involve in myoblast differentiation. They act by transactivate cathepsin B promotor activity and inducing its mRNA expression.
Similar to other E-box binding proteins, E47 also binds to the CANNTG sequence in the E-box. In homozygous E2A knock-out mice, B cells development stops before the DJ arrangement stage and the B cells fail to mature. E47 has been shown to bind either as heterodimer(with E12) or as homodimer(but weaker).
Researchers at the Medical School of Nanjing University found that the amplitude of FBXL3 (F-box/Leucine rich-repeat protein) is expressed via an E-box. They studied mice with FBXL3 deficiency and found that it regulates feedback loops in circadian rhythms by affecting circadian period length.
A study published April 4, 2013 by researchers at Harvard Medical School found that the nucleotides on either side of an E-box influences which transcription factors can bind to the E-box itself. These nucleotides determine the 3-D spatial arrangement of the DNA strand and restrict the size of binding transcription factors. The study also found differences in binding patterns between in vivo and in vitro strands.
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