Question

Using the components in the accompanying diagram, design regulatory modules (i.e., enhancersilencer modules) required for “your” gene to be expressed only in differentiating (early) and differentiated (late) liver cells. Answer the three questions presented below by describing the roles that activators, enhancers, repressors, silencers, pioneer factors, insulators, chromatin remodeling complexes, and chromatin readers, writers, and erasers will play in the regulations of expressions of your gene, that is, what factors will bind and be active in each case? Specify which transcription factors need to be pioneer factors.
1. How will the gene be activated in the proper cell type?
2. How will its expression be maintained?
3. How will expression be prevented in other cell types?

Polycomb complex Trithorax complex НАТ complex Reader H-acety modifiers Writer HDAC complex Eraser Insulator НЗК27me НЗК4тe НЗКОme Histone octomer RNA pol I remodeler SWISNF Complex Your gene Downstream Upstream Gene gene A Expressed everywhere B Expressed early in L, K, S. G C Expressed early in L, K, N D Expressed late in K,L ACT 1 Expressed everywhere 2 Expressed early in L, K, S, G 3 Expressed early in S, G 4 Expressed early in K, S 5 Expressed late in S, G REP - L Liver; K kidney; S spleen; G gall bladder; N neurons

Answer 1

1. In a proper cell a gene is activated by a series of activities that include the unwinding of the DNA at that position, the activation of the gene by removal of repressor or binding of activator at the promoter and binding of RNA polymerase to start transcription. Chromosome is made up of whole DNA sequence , coiled in a specific manner around the histone octamer proteins under normal conditions. When a cell needs a gene to be expressed, the first step is to unwind the DNA to free it from the highly coiled conformation so that transcription proteins can get access to the gene promoter. Histone acetyl transferases (HAT) are proteins that transfer an acetyl group to specific lysine residue on the histone protein, thereby neutralizing the positive charge of histone and weakening it's association with the negatively charged DNA. Chromatin remodeling is also carried out by a number of other histone modifications such as methylation, ubiquitination, phosphorylation etc. Specific lysine methylation such as H3K4 and H3K36 correspond to expression of gene. In eukaryotic system this remodeling complex is substituted by SWI/SNF complex that carry out the more or less same function. For developmental genes, trithorax complex protein help in post-translational modification of histones to help express genes of interest.

Once the gene promoter is free, activator proteins can bind enhancer regions (cis regulatory element on DNA) and help binding RNA polymerase through recruitment of required sigma factor. Thus the gene is activated.

2. Through chromatin remodeling are not irreversible, they can be retained for continual expression of genes. Dynamic remodeling doesn't work in developing embryo and in other cells where housekeeping genes are always needed to remain turned on. It is possible that Histone Deacetylases (HDAC) are somehow outnumbered or blocked in the site of gene expression that would otherwise remove the acetyl groups from the lysines of histone and promoting coiling of DNA around histone proteins. Apart from this chromatin accessability , the promoter region is kept activated by excess supply of activator proteins that outnumber the repressors in concentration and bind the enhancer regions thereby allowing RNA polymerase to bind and transcribe that portion of DNA repeatedly.

As in for housekeeping genes, the promoters are depleted of TATA box and the region is hugely dispersed over more than 100 bp sequence in contrast to other genes that don't need continued activation or expression. How this dispersed elements confers the constitutive activation, is still not clear.

3. Genes can be turned off by various ways. All cells in our body contain all the genes in the DNA content but the genes are expressed only in the relevant cells and remain turned off in other cell types. Chromatin remodeling complexes often provide means to deactivate gene expression by promoting tighter association between histone proteins and DNA. H3K9 and H3K27 methylation are correlated with the gene repression. Histone deacetylases (HDAC) remove acetyl groups and tighten the bond between DNA and histones thereby blocking the access of transcription factors .

Beyond the chromatin level, DNA contain cis-regulatory elements called insulators. Insulators make loops with DNA and block the enhancer from binding any activator protein. Insulators also create barrier between enhancer and promoter through looping and repress gene expression. Sometimes , trans regulatory proteins called repressors bind DNA promoter and in the absence of any repressor binding protein that could strip off the repressor from the promoter, the gene remain deactivated and does not get expressed.