Exercise 21 - Gene regulation: Human STX7
(a) Find the Location tab (Region in detail page) for the STX7 gene. Are there regulatory features in this gene region? If so, where in the gene do they appear?
(b) Click Configure this page and on the Regulatory features menu in the left hand side. Turn on Segmentation features for HUVEC, HeLa-S3, and HepG2 cell types. Do any of these cells show non-gene associated regulatory features in the STX7 region?
(c) Use Configure this page to add supporting data indicating open chromatin for HeLa-S3 cells. Are there sites enriched for marks of open chromatin (DNase1 and FAIRE) in HeLa cells at the 5’ end of STX7?
(d) Configure this page once again to add histone modification supporting data for the same cell type as above (e.g.HeLa-S3). Which ones are present at the 5’ end of STX7?
(e) Is there any data to support methylated CpG sites in this region (5’ end) of STX7 in B-cells?
(f) Create a Share link for this display. Email it to yourself then open the link.
Exercise 22 – Regulatory features in human
The HLA-DRB1 and HLA-DQA1 genes are part of the human major histocompatibility complex class II (MHC-II) region and are located about 44 kb from each other on chromosome 6. In the paper ‘The human major histocompatibility complex class II HLA-DRB1 and HLA-DQA1 genes are separated by a CTCF-binding enhancer-blocking element’ (Majumder et al J Biol Chem. 2006 Jul 7;281(27):18435-43) a region of high acetylation located in the intergenic sequences between HLA-DRB1 and HLA-DQA1 is described. This region, termed XL9, coincided with sequences that bound the insulator protein CCCTC-binding factor (CTCF). Majumder et al hypothesise that the XL9 region may have evolved to separate the transcriptional units of the HLA-DR and HLA-DQ genes.
(a) Go to the region from bp 32,540,000 to 32,620,000 on human chromosome 6
(b) Is there a regulatory feature annotated in the intergenic region between the HLA-DRB1 and HLA-DQA1 genes that has CTCF binding supporting data as (part of) its core evidence?
(c) Has the CTCF binding detected at this position been observed in all cell/tissue types analysed?
(d) Have a look at the Regulatory supporting evidence - Histones & Polymerases configuration matrix. For which cell/tissue type are the most histone acetylation data sets available? In this cell/tissue type, is the region that shows CTCF binding also a region of high acetylation, as found by Majumder et al?
Exercise 23– Methylation data in human
The human PDHA2 gene, that encodes for a subunit of the pyruvate dehydrogenase complex, is exclusively expressed in spermatogenic cells. In the paper ‘Human testis-specific PDHA2 gene: Methylation status of a CpG island in the open reading frame correlates with transcriptional Activity’ (Pinheiro et al Mol Genet Metab. 2010 Apr;99(4):425-30), two CpG islands in the PDHA2 gene are reported, one encompassing the core promoter region and extending into the open reading frame, the other exclusively located in the coding region. The latter CpG island was shown to be methylated in somatic tissues but demethylated in testicular germ cells and has therefore been proposed to play an important role in the tissue-specific expression of the PDHA2 gene.
(a) Find the PDHA2 gene for human and go to the Region in detail page. Zoom out one step, so that 5 kb around the PDHA2 gene is shown.
(b) Turn on the CpG islands track. There two CpG islands in the PDHA2 gene. Do they appear in this track? If not, why not? (Tip: turn on Display empty tracks to confirm that a track is on but has no data.)
(c) Confirm the existence of the two CpG islands using the EMBOSS program CpGPlot (http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html) on the sequence around the PDHA2 gene.
(d) Upload the CpG islands found by CpGPlot using Manage your data. Use BED format, which in its simplest form just consists of the chromosome and the start and end coordinates, separated by spaces (as an optional fourth field, you can add a name/description). The genomic start and end coordinates of the CpG islands can be calculated from the genomic start coordinate of the sequence on which the CpGPlot program was run and the relative location of the CpG islands on this sequence as given by the CpGPlot output.
(e) Create a link to allow you to show your new BED track to colleagues, compared to the %GC track.
(f) Are the two CpG islands methylated in somatic tissues but demethylated in sperm?