Question

8. Epigenetic modifications to DNA sequences and resulting alterations in chromatin structure can be analyzed by examining DNA methylation and histone modifications. To examine the methylation of a DNA sequence, you treat it with sodium bisulfite.  

a. (2 points) If your original DNA sequence is: 5’-ACAGTCCGTCGGAGCCTGCCAGTCGATCGCACCT-3’ and your sequence after sodium bisulfite treatment reads: 5’-ACAGTTCGTCGGAGCTTGTTAGTCGATCGCACTT-3’, which positions on the original DNA sequence are methylated? (Indicate methylations by circling the methylated nucleotides.)

b. (2 points) Draw the double-stranded DNA sequence after this original sequence is replicated. Be sure to indicate all methylated nucleotides on the old strand and the new strand

by circling them.

c. (3 points) How does DNA methylation affect chromatin structure and what does this do to gene expression?  Be specific.

d. (3 points) Describe how chromatin structure is altered in the process of X-inactivation and why the active X-chromosome is not inactivated. Include the functions of Jpx, Xist, Tsix, Xite, and describe what each of these gene products do. Be specific.

Answer 1

a

Sodium bisulfite methylates cytosine to form uracil. In DNA uracil is converted to thymine base as uracil is not a natural nucleotide in DNA. However, it will not affects the methylated cytosine of DNA. Therefore those cytosines are not changed after treatment with sodium bisulfite are present in methylated form. The highlighted cysteines are methylated because they are not changed to thiamine after treatment with sodium bisulfite.

5'- ACAGTCCGTCGGAGCCTGCCAGTCGATCGCACCT-3

b

The following strand is the original strand. Methylation sites have been shown in yellow.

5'- ACAGTCCGTCGGAGCCTGCCAGTCGATCGCACCT-3

The above strand after treatment with sodium bisulfite following strand will be generated. Those cytosines who were not methylated is converted to Thiamin. These are highlighted in blue.  

5’-ACAGTTCGTCGGAGCTTGTTAGTCGATCGCACTT-3’,

And after replication of the above strand following double-strand will be formed. The newly form strand will not have any methylated nucleotides. Therefore the duplex will be hemimethylated.

5’-ACAGTTCGTCGGAGCTTGTTAGTCGATCGCACTT-3’----Old strand methylated cytosine is highlighted

3'-TGTCAAGCAGCCTCGAACAATCAGCTAGCGTGAA-5'----New stand after replicated.

c

DNA methylation prevents methylation of Lys4 of histone H3, methylation of Lys9 of histone H3, and the deacetylation of histone H4. These modifications alter the structure of the chromosome which affects gene expression.

Gene expression gets suppressed in the presence of methylation. DNA methylation does this by inhibiting bind of transcription factors to the DNA and recruiting proteins involved in gene repression.

d

The answer is explained below