UBLCP1 Expression in Human B and T Cells

Emily Hinkle
November 29, 2018
Fall 2018
Dr. Ealy
Background

Some individuals who are immunosuppressed by human immunodeficiency virus (HIV)

infection and live in areas hyperendemic for tuberculosis (TB) seem to have a unique resistance

to developing active tuberculosis. The investigation of this phenomenon could be beneficial in

developing new treatments for TB in individuals who are not resistant to disease development. A

recent study which examined the incidence of TB in HIV positive individuals showed that the

minor allele of rs4912437 was correlated with resistance to TB development. Interestingly, a

gene in which this SNP was located, UBLCP1, was downregulated in TB resistant individuals

harboring the minor allele.1

The protein encoded by UBLCP1 is a 26S proteasome phosphatase, which serves to

regulate nuclear proteasome activity by dephosphorylating the 26S proteasome. UBLCP1

regulates the ubiquitin-proteasome system, which serves to degrade many proteins in the cell.

Deregulation of this system has been correlated to the development of many types of diseases,

including carotid stenosis, psoriasis, and platelet aggregation.2 The variants in introns of

UBLCP1 that the TB resistance study found to be correlated with TB development are at linkage

disequilibrium with variants in IL12B, a subunit of IL-12. The function of IL-12 is to cause the

“differentiation of naïve T cells into T helper 1 cells”. IL-12 has been shown to stop

mycobacterium tuberculosis bacteria from dividing and infecting the host. Variants in IL12B

have also been linked to carotid stenosis and psoriasis.1 IL12B and UBLCP1 were both shown to

have variants associated with the development of active tuberculosis. UBLCP1 was shown to be

downregulated in individuals harboring the minor allele and therefore resistant to TB infection.

One way that the gene could be downregulated is through different epigenetic events. The study

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of UBLCP1 and its epigenetic state in those who harbor the TB resistance allele could lead to

advanced knowledge in many diseases that the ubiquitin-proteasome system influences.

I propose that the epigenetic state of UBLCP1 is different in the B and T cells of

individuals who harbor the TB resistance allele. In order to test this, I will be looking at the

difference in UBLCP1 expression between individuals harboring the major and minor alleles. I

will also be testing the histone acetylation at H3K27 where the TB resistance allele is found. In

addition, I will test the DNA methylation state of the promoter for UBLCP1. If there is a

difference in the epigenetic state of B and T cells in individuals that harbor the resistance allele,

we could potentially discover the ramifications of methylation or acetylation at different sites.

Specific Aims

Hypothesis: The expression of the UBLCP1 gene in the B and T cells harboring the

rs4912437 SNP minor allele is downregulated as a result of the epigenetic state.

Aim 1 will test the difference in histone markers between individuals who have the

rs4912437 minor allele as opposed to the major allele. It is expected that there would be less

acetylation of histone markers in individuals who have the minor allele resulting in lower

UBLCP1 expression. Additionally, the histone acetylation at H3K27 will be tested. Presence of

decreased acetylation of histone markers and decreased H3K27 histone acetylation would

suggest heterochromatin formation at the UBLCP1 locus.

Aim 2 will test the methylation state of the DNA at the promoter of UBLCP1. Potential

variation in UBLCP1 expression between those who have shown resistance to disease and those

who have not will be beneficial in determining the role of this gene in disease development. It is

expected that the UBLCP1 gene will have decreased expression in the B and T cells harboring

the minor allele which has been shown to lend resistance to disease. It is also expected that the

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DNA at the promoter of UBLCP1 will have increased methylation in individuals harboring the

minor allele.

Experimental Design

To test Aim 1, the expression of UBLCP1 will be tested in B and T cells of individuals

harboring the minor allele as well as those with the major allele using a quantitative reverse

transcription PCR of UBLCP1. These will be compared to determine if there is variation in the

expression between these groups. Next, the acetylation of the histone proteins in the genome

surrounding the UBLCP1 gene will be tested in each group using the Chromatin

Immunoprecipitation (ChIP) method. The acetylated histones will be immunoprecipitated using

an antibody specific to acetylated histone H3. Quantitative reverse transcription PCR will be

performed for the promoter of the UBLCP1 gene. Comparison between cells with the minor

allele at rs4912437 and the major allele will be performed to identify differences in the

acetylation state of the markers in each group.

To test the H3K27 histone, the H3K27 acetylated histones and the associated DNA will

be immunoprecipitated also using the ChIP method. Primers will be designed for the promoter of

UBLCP1 and quantitative reverse transcription PCR will be performed. These samples will be

analyzed using quantitative reverse transcription PCR. This will quantitatively show the

acetylation of DNA at H3K27, indicating its level of expression in each group.

To test Aim 2, UBLCP1 expression in the B and T cells of individuals who harbor the

minor allele will be tested as well as those of individuals who harbor the major allele. This will

be done using quantitative reverse transcription PCR of UBLCP1. Next, bisulfite sequencing will

be performed at the promoter region of UBLCP1 to test the methylation state of this DNA. B and

T cells from both groups will also be bisulfite-treated, and the promoter region amplified by

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PCR, and then sequenced. The untreated sequence and bisulfite treated sequences from each

group will be compared. The cytosines which have been transformed to tyrosine in the bisulfite

treated group will be considered to have been unmethylated. Similarly, the cytosines from the

bisulfite treated group which have remained cytosine will be considered to have been

methylated.

Expected Results:

It is expected that UBLCP1 will have decreased expression in the B and T cells of

individuals harboring the minor allele in both tests. If UBLCP1 were decreased in these

individuals, the 26S proteasome could not be dephosphorylated as often and as a result, would

degrade less proteins in the cell. This could be a contributing factor in the resistance to TB as

well as other diseases as one of these proteins could increase immune response to certain

infections. In Aim 1, it is expected that there will be decreased histone acetylation in the minor

allele as well as decreased acetylation of the H3K27 histone associated DNA. The presence of

both marks would indicate the formation of heterochromatin, resulting in decreased UBLCP1

expression in individuals harboring the minor allele. It is also expected that there will be

increased histone acetylation in individuals harboring the major allele as well as increased

acetylation of the H3K27 histone associated DNA, indicating the formation of euchromatin. In

Aim 2, it is expected that there will be increased methylation of the promoter in the individuals

who harbor the minor allele.3 This will be shown in the presence of remaining cytosines after

bisulfite treatment. Similarly, it is expected that there will be decreased methylation of the

promoter in individuals harboring the major allele.

If UBLCP1 has increased expression in individuals who harbor the minor allele, it could

indicate that something else is contributing to these individuals’ increased resistance to TB and

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possibly other diseases. This could contribute to future research focused on the other diseases

UBLCP1 has been shown to play a role in, such as carotid stenosis and psoriasis, by providing

increased information on the expression of UBLCP1. By gathering information on the expression

of this gene, future studies on the incidence of tuberculosis in HIV individuals will be able to use

this to determine the cause of increased resistance to TB in individuals harboring the minor

allele. Further, future studies on other factors in disease development could use this data to

determine other processes that UBLCP1 could be contributing to. It is also possible that UBLCP1

is expressed differently in other tissues besides B and T cells, so this work could provide a

framework for studies of this type.

If UBLCP1 is not expressed differently between the 2 groups, but there is a difference in

the epigenetics, this could contribute to the understanding of UBLCP1’s natural state and the

dephosphorylation of the 26S proteasome. In the study of many diseases that are influenced by

the increase or decrease in activity of the proteasome such as Alzheimer’s, Parkinson’s, and

HTLV, UBLCP1 epigenetics could be playing a role.4 If there is a difference in the expression,

but no difference in the epigenetic marks, there could be future research done on what is causing

the difference in expression which could lead to better understanding of UBLCP1’s role in

regulation of the 26S proteasome. If there is no difference in the expression or epigenetic marks,

this would imply that another modification could be causing the variation in resistance to TB.

This could be a mutation to the UBLCP1 gene or a post-translational modification or lack

thereof. This could cause future research in this area, such as the modifications that UBLCP1

undergoes in test individuals.

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 References

1. Sobota, R. S., Stein, C. M., et al. (2016). “A Locus at 5q33.3 Confers Resistance to
Tuberculosis in Highly Susceptible Individuals”. American Journal of Human
Genetics. 98(3): 514-524.
2. Guo, X., Engel, J. L., et al. (2011). “UBLCP1 is a 26S proteasome phosphatase that
regulates nuclear proteasome activity”. Proceedings of the National Academy of Sciences
of the United States of America. 108(46): 18649-54.
3. Guastafierro, T., Bacalini, M. G., et al. (2017). “Genome-wide DNA methylation analysis
in blood cells from patients with Werner syndrome”. Clinical Epigenetics. 9: 92.
4. Burkhardt Dahlmann. (2007). Role of Proteasomes in Disease. BMC Biochemistry. 8
Suppl 1 (Suppl 1), S3.