Question

5. With regard to eukaryotic chromatin, what is a DNase hypersensitive site? What types of DNA sequences tend to be located at these sites? How would you demonstrate experimentally that a specific sequence is located with a hypersensitive site/region? Describe and provide hypothetical results. What is an LCR?

Answer 1

What is a DNase hypersensitive site?

DNase hypersensitive site is a short region of chromatin which has high sensitivity to cleavage by DNase I and various other nucleases (DNase II and micrococcal nucleases). In a hypersensitive site, the nucleosomal structure is less compacted, increasing the availability of the DNA to binding by proteins, such as transcription factors and DNase I. These sites account for many inherited tendencies.

What types of DNA sequences tend to be located at these sites?

In these specific regions of the genome, chromatin has lost its condensed structure, exposing the DNA and making it accessible. This raises the availability of DNA to degradation by enzymes, such as DNase I. As the most accessible regions of chromatin to non-histone DNA-binding proteins, DNase I hypersensitive sites generally denote those DNA sequences which have important functions in the nucleus.

How would you demonstrate experimentally that a specific sequence is located with a hypersensitive site/region?

​ DNaseI hypersensitivity assays can experimentally demonstrate this. This  DNaseI hypersensitivity assay involves treating chromatin briefly and with limiting amounts of DNaseI will cleave exposed hypersensitive sites. It is important to note that longer digestions or greater amounts of the enzyme will cleave the DNA of active genes at multiple sites, referred to as general DNaseI sensitivity.  Detection of the hypersensitive sites thus requires treating nuclei, containing intact chromatin, with limited amounts of DNaseI for a short period such that partial digestion is achieved, and then halting the digestion to prevent further DNA degradation. To map the cleavage sites, the DNA is isolated, digested with an appropriate restriction endonuclease, and analyzed by traditional Southern blotting. The hypersensitive sites are revealed by indirect end-labeling, using a short probe complementary to sequences near, or at the end of, the predicted restriction fragment. This produces a series of fragments and permits mapping of the cleavage sites to specific regions.

What is an LCR?

LCR is the locus control region (LCR). It is a long-range cis-regulatory element that enhances expression of linked genes at distal chromatin sites. It functions in a copy number-dependent manner and is tissue-specific, as seen in the selective expression of β-globin genes in erythroid cells. Expression levels of genes can be modified by the LCR and gene-proximal elements, such as promoters, enhancers, and silencers. The LCR functions by recruiting chromatin-modifying, coactivator, and transcription complexes.

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