DREB (Dehydration Responsive Element Binding) | A sub-family of Transcriptional Factors

  DREB 

(Dehydration Responsive Element Binding)

Background of DREB transcription factors.

DREB (Dehydration Responsive Element Binding) is the transcriptional factor that activates the genes in stress conditions. It regulates the genes under both biotic and abiotic stress stimuli. 

DREB is the subfamily of ERF (Ethylene Response Factor).  It was discovered by a team of many researchers. Kazuko Yamaguchi Shinozaki, Kazuo Shinozaki, Jian Kang Zuo are some of them. They all were working on the Arabidopsis thaliana plant at the University of Tokyo in Japan which then led to the discovery of dehydration dehydration-responsive element binding protein.

Kazuko Yamaguchi Shinozaki and others were plant molecular scientists and biologists. They all worked on the genes, that expressed in stressed conditions. One of their experiment was on the Aradiposis plant. They tried to compare different genes of this plant in both stressed and un-stressed conditions and found that DREB1A is one of the transcriptional factors that is involved in the regulation of genes that deal with stressed conditions.

Now, how do they compare the different genes of Aradiposis in both stressed and unstressed conditions?

They were able to compare the gene expression in both stressed and unstressed conditions with the help of a technique named Differential Display Reverse Transcription-polymerase chain reaction (DDRT-PCR). This is a special technique used in molecular biology to compare the expression of several genes under stressed and unstressed conditions.

DDRT-PCR Technique.

The technique of Differential Display Reverse Transcription-PCR has the following steps:

The term reverse transcription in DDRT-PCR shows that the process of reverse transcription is essential to compare the expression of genes in different conditions. So we have to understand the technique of the reverse transcription process first.

• Reverse transcription is the process in which complementary DNA (cDNA) is formed from single-stranded messenger RNA (mRNA) with the help of a reverse transcriptase enzyme.
• For the preparation of cDNA, we have to isolate RNA from cells.
• The RNA isolation can be done by cell culture technique or tissue dissection method.
• Once the RNA is isolated from the cell it is important to stabilize it by using stabilizer agents like RNAlater, RNAsnap, trizon, etc. 
• These stabilizer agents help to prevent the RNA from degradation.
• The isolated RNA is also treated with DNA-digesting enzymes (DNAase) and protein-digesting enzymes (proteinase).
• The isolated RNA is purified by using the column purification method. This will remove all the impurities from the RNA.
• At last, the isolated RNA is passed from spectro-photometry. This is done to check whether the isolated RNA is useful for further experiments or not.
• After a pure sample of RNA is isolated from the cell, it is treated with reverse transcriptase enzyme and primer.
• The reverse transcriptase enzyme forms a single-stranded cDNA.
• After single-stranded cDNA is formed, it is treated with DNA polymerase enzyme and a second primer.
• DNA polymerase enzyme converts the single-stranded cDNA into double-stranded cDNA which is more stable.
• This double-stranded cDNA is also purified by using the column chromatography technique. 

With the help of reverse transcription, we can prepare copies of complementary DNA (cDNA). These copies can be single-stranded or may be double-stranded. It all depends on the protocols of the experiments that one is performing. However, the copies of this cDNA are then inserted into a PCR machine where we can get multiple copies of this cDNA.

PCR (Polymerase Chain Reaction).

• Set a polymerase chain reaction (or PCR) for this double-stranded cDNA.
• In PCR, we first break the double-stranded DNA into two molecules of single-stranded DNA by increasing the temperature (up to 90c).
• Then decrease the temperature and provide polymerase enzyme and primers to these two strands of single-stranded DNA molecule.
• Add many small single-stranded nucleotides of DNA. 
• These nucleotides are the group of sets of ddATP,ddCTP, ddTTP, and ddGTP. Here “dd” means “di-deoxy” which means deficiency of two oxygens.
• Primers used the nucleotides and with the help of the DNA polymerase enzyme, it formed two molecules of double-stranded DNA.
• Here the first PCR cycle is completed and the amount of cDNA is doubled.

In the first cycle of PCR, the amount of DNA is double which means our one molecule of double-stranded DNA now becomes two molecules of double-stranded DNA. In the second cycle of PCR, the number of molecules of DNA will be four. In the same way, after completing the third cycle of PCR, we will get eight molecules of this double-stranded cDNA and this process continues. Repeat this cycle about 30-40 times so that we will get a healthy concentration of cDNA. After getting a healthy concentration of DNA, we subjected this cDNA to gel electrophoresis.

• The sample of cDNA was then subjected to gel electrophoresis. 
• The principle of gel electrophoresis is based on the thing that long strands of DNA move slower on gel electrophoresis while short strands of DNA move faster on it.
• We will get a band pattern of different-sized DNA molecules that traveled on gel electrophoresis.
• This difference in the band pattern indicates the different gene expression in different conditions by different cells.

Gene Regulation by DREB Transcription Factors.

In this way, they were able to detect the gene expression in both stressed and un-stressed conditions. The transcriptional factors control the regulation of genes and decide which genes need to be expressed according to conditions and organism or cell needs. They controlled the process of transcription, which is the formation of mRNA from DNA.

Normally Genes are classified into two types i.e. structural genes and regulatory genes. Structural genes form structural proteins like proteins in our hair, nails, muscles, bones, etc. While the regulatory genes controlled the regulation of genes. In simple words, we can say that regulatory genes decide whether the protein is formed in the cell or not. It turns on/off of the genes. 

There are well-controlled and very complicated mechanisms present in the cells that control the expression of genes and the transcription factors are part of the process of gene regulation.

DREB (Dehydration Responsive Element Binding) is the group of transcriptional factors that regulates the expression of genes. GhBRED2 is an example of a transcriptional factor of cotton that controls the expression of genes that are responsible for making proteins like aquaporin, heat-shock proteins, chaperone proteins, and antioxidant enzymes. 

BRED transcriptional factors of the cotton plant (Gossypium hirsutum) also include GhBRED1, GhBRED2, GhBRED3, GhBRED4, etc. These are some transcriptional factors that express the genes in stress conditions like drought stress, light, temperature stress, etc. in cotton plants. These transcriptional factors also have a role in the development and production of fiber in cotton.

Here we have to understand how these transcritional factors help in the regulation of genes.

• The first thing that transcriptional factors do is to identify its promotor site on DNA.
• Once they find the promotor region on DNA, it binds with DNA.
• DNA is mostly wrapped around histone protein which is also called methylation of DNA because methyl groups are normally responsible for this wrapping. 
• DNA unwraps with the help of an acetyl group and it is called acetylation of DNA. So it is important to unwrap the DNA first before any transcriptional factor binds with DNA.
• The transcriptional factor after binding with DNA, recruits RNA-polymerase with it to form mRNA from DNA.
• It forms a transcription initiation complex (transcriptional factor + RNA polymerase) to make mRNA from DNA.
• The transcription factor like GhBRED2 expresses its genes to form mRNA (transcription process). 
• Once mRNA is formed, it forms proteins (translation process).
• Once the desired quantity of protein is formed by the cell, then the transcription factor (e.g. GhBRED) turns off that gene by adding a repressor with it.

 In this way, transcription factors perform their functions and regulate the gene expression. It is also important to understand that transcriptional factors are only produced when they are needed. Like GhBRED2 is only produced in cotton under stress conditions because it regulates the genes of cotton that are responsible for stress conditions.