The that the transcript is an unprocessed

The initial
outlook of the KRT1 gene is that it is inactive for transcription in K562
cells. This conclusion is supported by the epigenetic profiles, in Figure 3, of
the KRT1 gene.

The present
limited peaks of H3K4m1 show that the gene has potential of being transcribed,
but not enough to be considered significant. This is supported by the
comparison of activity between the H3K4m1 marker for the KRT1 gene and the same
marker in the GATA3 gene; both limited but show potential for transcription.

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Alongside
this the low activity of the H3K4m2 and H3K4m3 show that the gene is not being
transcribed. This is also shown in the comparison between H3K9ac and H3K9m3
peaks, (despite being reduced), the peaks for H3K9m3 are more significant than
that of acetylation (H3K9ac). Therefore, it can be indicated that there is more
potential for repression than activation. This is further supported by a comparison
between H3K27ac and H3K27m3, where there are more significant peaks of
repression on the tri-methylated mark, towards the end of the transcript, than
the acetylated mark. As a result, the chromatin is not being made accessible to
transcription factors, so the gene remains inactive. (Chen, D., et al. 2005).

In addition to this, there is a lack
of significant peaks for the Pol2 mark along the gene. This indicating that no
transcription is taking place across the gene transcript, therefore remains
inactive. Also, there is no presence of a CpG marker at the beginning of the
gene, normally indicative of a promoter region. Potential reasons for this is
that the gene does not contain a promoter region so will not appear on the ChIP
analysis, or that the transcript is an unprocessed pseudogene for the KRT1
gene; where they have lost some functionality relative to the complete gene.
(Tutar, Y. 2012). Lastly, the lack of a CpG island results in the nucleosomes
being evenly spaced along the gene, implying that it is heterochromatin and the
gene is inactive. In support of this, the DNase1 activity is at a continuously
low level throughout, implying there is little space for it to cut and
therefore results in the chromatin type to be heterochromatin (tightly packed).
Therefore, the KRT1 gene is inactive in K562 cells. The KRT1 gene is
formed by type II cytokeratins set in pairs in a heterotypic chain. This gene
encodes for Keratins, specifically Keratin 1, which are tough fibrous proteins
that form the structural framework of cells that make up the skin, hair and
nails. This gene is therefore considered a tissue specific gene. (NCBI KRT1
entry. 2017). The
initial outlook of the GATA3 gene, located on chromosome 10, is that it is
inactive for transcription within K562 cells. This conclusion is supported by
the overall epigenetic profiles shown in Figure 2.The
present but limited peaks of H3K4m1 show that the gene has potential to be
transcribed, but not high enough to be considered significant. This can result
in the displacement of transcription factors from their cognate sites, not allowing
transcription. (Slattery, M., et al.2014).
Alongside this the low activity of H3K4m2 and H3K4m3 show that the gene is not
being transcribed, or poised for transcription. Similarly, the comparison
between H3K9ac and H3K9m3 peak levels, despite being reduced, show that the
peaks for methylation are more significant than acetylation. Therefore, there
is more potential for repression than activation. This is supported by a comparison
between H3K27ac and H3K27m3, whereby there are more significant peaks of
repression on the tri-methylated mark than the acetylated mark. As a result,
the chromatin is not being made accessible to transcription factors, so the
gene remains inactive. In addition to this, there is a lack
of significant peaks for the Pol2 mark along the gene. This is indicative of no
transcription, because it not only requires RNA Pol2 but also the binding of
transcription factors. (Nikolov, D.B. 2007). Also, the presence of a CpG marker is
indicative of a promoter region, implying that the gene has the potential to be
transcribed. This is supported by the marker for nucleosome activity i.e. K562
Sig, showing that, at the beginning, the nucleosomes are not evenly spaced but
become more regularly spaced. This could imply that they have been pushed aside
to make space for the transcriptional machinery, due to there being slight
potential for transcription.

Finally,
the low level peaks for DNase1 activity indicate that there is little space
within the chromatin for it to cut, implying the chromatin type to be heterochromatin.
Therefore due to the condense packing of the chromatin, no transcriptional
machinery can reach the site to initiate transcription- indicating the gene to
be inactive. This could be a result of the GATA3 gene itself being more heavily
involved (mutated) in other cancers such as breast cancer (luminal A type)
(Takaku, M., et al. 2015). The GATA3 gene
encodes transcription factor GATA3, which is an important regulator of T helper
cell development, promotion of the secretion of interleukins and
differentiation of epithelial cells. (Ho I.C., et al. 2009). This gene is considered tissue specific because the
expression is limited to the immune system and epithelial cells. (Asnagli, H., et al. 2002).The
initial outlook of the GAPDH gene, located on chromosome 12, is that it is
actively involved in the transcription process within K562 cells. This
conclusion is supported by the overall epigenetic profiles shown in Figure 1 of
the GAPDH gene. The
present, but smaller, peaks of H3K4m1 show that the gene has the possibility of
being transcribed as it represents a marker of primed enhancer activation by
modulating nucleosome mobility. This therefore results in a more dynamic
chromatin structure, facilitating transcription factor accessibility. (Calo,
E., Wysocka, J. 2013). Alongside this the high activity of H3K4me2, which
passes the threshold unit, shows that the gene is being actively transcribed
and potentially containing transcription factor binding regions to indicate
this activity. Similarly, the high activity of H3K4me3 above the threshold unit
indicates active transcription of the gene; this epigenetic profile can be
considered as a marker of active promoters. Furthermore, the comparison between the
activity of the H3K9ac and the H3K9m3 marker can indicate transcription of the
gene. The high, above threshold, peaks for H3K9ac show that the histone is
being acetylated; this making the chromatin more accessible to transcription
factors, as compared to the very low peaks of H3K9m3 whose function is to
repress transcription. This is also shown in epigenetic markers H3K27ac and
H3K27m3. (Becker, J., et al. 2016). In
addition to this, peaks in the Pol2 marker clearly indicate gene activation of
transcription as it is strongly linked with DNA replication. Also, the presence
of the CpG marker at the beginning of the gene is indicative of a promoter
region, implying that the gene can be transcribed.

Finally, from the two high peaks indicative of
DNase1 activity, K562 DS and K562 OS, and the irregular positions for
nucleosomes, K562 Sig, indicate that the chromatin type is Euchromatin-
therefore the gene is active. This is due to space being readily made for the
transcriptional machinery, which cannot happen if it were heterochromatin. (Becker,
J., et al. 2016). Therefore, the
GAPDH gene is active in K562 cells.The
K-562 cell line was derived from a Chronic Myeloid Leukaemia (CML) patient at
the terminal stage of the disease. (Bassat B., et al. 1976). Genes within this cell line can be considered as housekeeping
genes or tissue specific and their
expression can be regulated epigenetically. Glyceraldehyde
3-Phosphate Dehydrogenase (GAPDH)

GAPDH
is a commonly used housekeeping gene. It is important for
energy metabolism and the production of ATP through anaerobic glycolysis. It is
also involved in transcription activation, initiation of apoptosis,
tumorigenesis and normalisation of gene expression. (Barber R.D., et al. 2005). 

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