1. The Roles of ELL2 and ELL3 in Plasma Cell Development
Shane McKeon and Aleksandra Basina Christine Milcarek
First Experiences in Research, Dietrich School of Arts & Sciences, University of Pittsburgh
Abstract
The Lenti Virus
DiscussionIdentifying ELL2 and ELL3
Acknowledgements
B cells express immunoglobulin (Ig) molecules composed of heavy and light chains on their surfaces; after
antigen or LPS stimulation they differentiate into antibody secreting plasma cells (ASCs) that make and secrete large
amounts of the Ig protein. One of the primary processes to influence the shift to Ig secretion is differential Ig heavy
chain RNA polyadenylation and splicing. We showed in unpublished data that the small nuclear RNAs important for
splicing are reduced in ASCs relative to B cells (Carew & Smith, personal communication). Previous studies showed
that (a.) the transcription elongation factor ELL2 is induced with differentiation to ASCs, (b.) conditional deletion of
ELL2 results in decreased Ig heavy chain secretory-specific mRNA production and diminished Ig secretion in the whole
animal and (c.) after LPS stimulation of B cells ELL3 decreases and ELL1 is unchanged (1). The ELL3 and ELL1 and
2 proteins are closely related in structure but ELL3 lacks the interior ~100 amino acids (2). The ELL proteins have been
shown to be involved in at least two transcription elongation complexes, the little elongation complex (LEC) involved
with small nuclear RNAs, and the super elongation complex (SEC) important for the bulk of genes transcribed in
Drosophila and human cancer cell lines (3). However, the differential involvement of ELL2 versus ELL3 was not
explored. We then hypothesized that the transition in expression of ELL3 to ELL2 is important in the development of B
cells into ASCs, and would influence both Ig secretory mRNA expression and small RNA synthesis. The Milcarek
Laboratory is using Quantitative PCR (QPCR), western blots and complementation of defects in knockout mice to
display the relationships. The research this semester was primarily focused on seeing whether or not ELL1, 2 and 3 had
similar functions. To do so, we first needed to test our cell lines to make sure we had the correct antibodies, and grow a
virus that could be used to correct deficiencies in the immunoglobulin secretion abilities of splenic B cells from ELL2
conditional knockout mice.
We began with two cell lines, MM1.S containing only ELL2, and SKW6.4 containing only ELL3. In order to
provide concrete support that these cells line expressed the desired proteins we ran a QPCR which showed that SKW6.4
does indeed contain ELL3 while MM1.S does not (Figure 1). Furthermore, a western blot was run using antibodies
complimentary to ELL2. Once the blot was transferred to a membrane and imaged with chemiluminescence, it was clear
that MM1.S contains ELL2, while SKW6.4 did not. (Figure 2)
Moving forward, Lenti virus DNA will be packaged and placed into knockout mice cells (contains no ELL2) and wild
type mice cells (contains ELL2). The cells without ELL2 should produce no immunoglobulin secretion as their immune
system has been compromised. ELL1, ELL2 and ELL3 will then be individually added to the knockout cells. It is
expected that ELL2 will correct this defect, however, the main purpose of this experiment is to see if ELL1 and/or ELL3
will also correct the defect. If so, it will prove that ELL1 and/or ELL3 have similar if not the same function within the
immune system. Future directions will involve characterizations of the LECs and SECs for B versus ASCs.
We would like to acknowledge Christine Milcarek for guiding us through this research and supporting our
love for research. Also we would like to thank FER for this experience.
Background Information
When B cells differentiate into antibody secreting cells many genes are
affected but little information is known. In a previous study, Dr.
Milcarek found that there were changes in the expression of small
nuclear RNA; an important component in splicing out introns from pre-
RNA to form mature mRNA. Additionally, it was found that ELL3
levels are diminished after stimulation from B cells to ASC. As seen in
the figure to the right, ELL3 is much lower in ASCs than B cells, while
ELL2 is much lower in B cells than ASCs. This information led to
looking into how snRNA is controlled during the transition from B cells
to ASCs.
It was hypothesized that ELL3 regulates the formation of SECs and
LECs in B cells while ELL2 drives the formation of the SECs over the
LECs when producing antibodies. It is this switch that benefits mRNA
instead of snRNA.
To test the hypothesis, we wanted to see if the down regulation of snRNA was
linked to the down regulation of ELL3. Naïve splenic cells will be stimulated
with LPS and then treated with inhibitors of protein synthesis or RNA
synthesis and the production of ELL3 and snRNA will be monitored. Prior to
doing so, the research mainly focused on identifying which cell lines contained
ELL2 vs ELL3 so that we could be sure we had the correct genes.
Additionally, we decided to grow a lenti virus that would be placed into ELL2
knockout splenic B cells. We would then inject the ELL proteins one at a time
and see if one, or all could correct the deficiency, by monitoring the
immunoglobulin secretion.
The image to the left shows the similarities and differences between all three
ELL proteins on a molecular level. As you can see most of ELL3’s DNA
sequence is unknown and the parts that are shown are similar to the other ELL
genes, suggesting a similarity between the genes functions.
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To obtain a virus to transfect into the splenic B cells we ordered bacteria that
contained a lenti virus, but needed to test if the sample actually contained the
desired virus. The sample came with a plasmid map (as seen to the left) , that
showed what enzymes would cut the plasmid, and at what locations. For our
purposes we choose three enzymes, EcoR1, Sac1 and Sal1. These enzymes
were predicted to produce segments of the plasmid at 6973 and 3401 for
EcoR1, 2960, 191, 7223 for Sac1 and 1618, 2185, 6571 for Sal1. To test if our
lenti virus would be cut in the appropriate locations, we mixed the enzymes
with their complementary buffers and the DNA and ran each enzyme on a DNA
gel , with three different samples of the lenti virus. In the gels below, lanes 6,
12, and 16 contain the lenti virus uncut, so serve as a control.
Lanes 7, 13, and 17 contain the lenti virus cut
with the EcoR1 enzyme and came out to be
around 6000 kDa, as expected.
Lanes 8, 14,and 18 contain the Sac1 enzyme and
as seen in lanes 14 and 18, two bands appeared,
one around 7000 kDa and another around 3000
kDa. Additionally, lane 8 contains the band at
7000 kDa and another very faint band around
200 kDa. All three of these bands were expected
when cutting the plasmid with sac1.
Lanes 9, 15 and 19 contain the lentu virus cut
with Sal1 and all three lanes contain three bands
around the 6000 kDa mark, the 2000 kDa and
the 1000 kDa mark. All three values are
consistent with the predicted values provided by
the plasmid map.
In order to confirm that the MM1.S cell line contained ELL2 while the SKW6.4
cell line did not, we ran a protein gel and western blot. Because the MM1.S cells
were ASCs we expected a much large abundance in ELL2 than in the B cells
(SKW6.4). In order to test the results the gel was transferred to a western blot and
coated with a primary antibody that was complementary for ELL2 overnight. It
was then washed and a anti- ELL2 secondary antibody was placed on the
membrane. We were then able to expose the membrane to chemiluminescence
and received the image to the left.
The expected molecular weight of ELL2 is 72 kDa. As seen in the image there
are thick bands within the ASC lanes that are near 72 kDa, as seen on the protein
ladder next to the membrane image. Additionally, there are no bands around 72
kDa under the B cell lanes, proving that ELL2 is expressed in ASCs but not B
cells.
PCR is typically used to amplify a specific gene, or portion of gene, so that we can
study the function of that gene or gene region. In our experiments, we amplify the
lentiviral DNA that contains a vector with the gene eGFP (green fluorescent protein)
as well as ELL3. Primers are used to flank the region you want to amplify. Each
primer will amplify the gene sequence on both strands, creating a double-stranded
gene product. The primers we used are represented at the figure below.
The PCR process follows 3 steps. Denaturation step: First, you heat the DNA to a
high temperature (95 °C) so that the two strands of genomic DNA, and later PCR
DNA, separate. Annealing step: Second, you reduce the temperature (around 60 °C)
so that DNA primers bind to either end of the template that you want to amplify. It
is important that you have two primers, one to bind
to each strand of DNA. Extension Step: Third, you
raise the temperature to about 70 °C to activate a
DNA polymerase and elongate the primer with
respect to the template strand.
Over the course of the semester in the Milcarek lab, we were
ultimately able to lay the ground work for further testing of whether
genes ELL1 and ELL3 have similar function in the immune response
to that of the gene ELL2.
We were able to ensure that the MM1.S cell line contained only
ELL2 and the SKW6.4 cell line contained only ELL3 through
quantitative PCR analysis as well as western blots using antibodies
known to be complimentary to ELL2 (and therefore able to detect its
presence). In addition, we were able to confirm that the lenti viral
vector containing our genes of interest were present in the bacterial
host, and therefore could be used to transfect into B cells of
conditional knockout mice.
References
1. Park, K. S., Bayles, I., Szlachta-McGinn, A., Paul, J., Boiko, J., Santos, P., Liu, J., Wang, Z., Borghesi, L.,
and Milcarek, C. (2014) J. Immunol. 193, 4663-4674
2. Miller, T., Williams, K., Johnstone, R. W., and Shilatifard, A. (2000) J. Biol. Chem. 275, 32052-32056
3. Luo, Z., Lin, C., and Shilatifard, A. (2012) Nat Rev Mol Cell Biol 13, 543-547
4. Sage M. Smith, Nolan T. Carew, and Christine Milcarek Department of Immunology, University of
Pittsburgh, School of Medicine, Pittsburgh, PA 15261, United States
Recall that B cells differentiate into antibody secreting plasma cells (ASCs) upon stimulation by an antigen or
lipopolysaccharide (LPS). ASCs make and secrete vast amounts of immunoglobulin molecules (Ig), and that this
is influenced by differential IG heavy chain RNA polyadenylation and splicing.
Also recall that ELL2 is the only gene of the three ELL genes that has previously been shown to act directly on
immunoglobulin genes. It has so far been established that ELL2 is induced at least 10-fold in antibody secreting
cells in comparison to B cells, while ELL1 expression decreases slightly and ELL3 expression decreases
tremendously. In turn, such changes cause the RNA polymerase II in antibody secreting cells to take on different
properties, ultimately resulting in changes concerning RNA processing and modification of histones (4).
Continued research will focus on the role of ELL1 and ELL3 and whether they may substitute ELL2 in such
functions.
In the future, the lenti virus containing the gene of
interest will be transfected into cells of conditional
knockout mice. This will test whether ELL1 and/or
ELL3 can have similar functionality as that
exhibited by ELL2. In addition, further research
may lead to characterization and analysis of the
long elongation complexes (LECs) and the short
elongation complexes (SECs) present in B cells as
opposed to in ASCs.