CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1
A thesis submitted in partial fulfillment of the requirements
For the degree of
Master of Science in Biology
By
Loni M Hands
August 2016
Copyright by Loni Hands 2016
ii The thesis of Loni Hands is approved by:
_____________________________________________
Kerry K. Cooper, Ph.D.
_______________
Date
_____________________________________________
Rheem D. Medh, Ph.D.
_______________
Date
_____________________________________________
Cheryl L. Van Buskirk, Ph.D.
_______________
Date
_____________________________________________
Cindy S. Malone, Ph.D., Chair
_______________
Date
California State University, Northridge
iii ACKNOWLEDGMENTS
I would like to thank my P.I., Dr. Cindy Malone, for her constant, strong support
throughout my time as a masters student.
I would like to thank my committee for their help and challenging questions
during my proposal and defense.
I would like to thank Dr. Daniel Odom for always being available to talk science,
reason through protocols, and help me with my lab techniques.
Finally, I would like to thank my family for always supporting me no matter what.
iv TABLE OF CONTENTS
Signature Page
iii
Acknowledgments
iv
Abstract
vii
Chapter 1: Introduction
1
1.1 Importance of gene regulation
1
1.2 Consequences of gene dysregulation
1
1.3 Gene regulation – Overview
2
1.4 Gene regulation – Core promoters and transcription initiation
3
1.5 Gene regulation – Proximal promoter elements (PPEs)
5
1.6 Gene regulation – Enhancers and silencers
6
1.7 Gene expression in mantle cell lymphoma vs. small lymphocytic
lymphoma
8
1.8 Nucleosome assembly protein 1-like 1 (NAP1L1)
9
1.9 Characterizing the NAP1L1 promoter region and enhancer element
Chapter 2: Methods
10
12
2.1 Cloning the NAP1L1 promoter region into pGL3 Basic
12
2.2 Cloning a larger fragment of the NAP1L1 promoter region into pGL3
Basic
14
2.3 Cloning a region of NAP1L1’s intron 1 into pGL3N-1273 and pGL3
Basic
16
2.4 Boiling lysis plasmid purification
17
2.5 Restriction digests
18
2.6 Transcription factor consensus sequence identification
18
2.7 Designing initial deletion constructs of the NAP1L1 promoter region
19
2.8 Designing smaller deletion constructs of the NAP1L1 promoter region
21
2.9 Mutating the myc binding site within the first intron of NAP1L1
24
2.10 Transfection of promoter constructs into human embryonic kidney
(HEK293T) cells
27
2.11 Harvesting transiently transfected HEK293T cells
28
2.12 Dual-Luciferase Assay of transfected pGL3 constructs
29
v Chapter 3: Results
30
3.1 Bioinformatics analyses of the NAP1L1 promoter region
30
3.2 Cloning the NAP1L1 promoter region into pGL3 Basic
32
3.3 Removing the intron from pGL3 Basic containing the NAP1L1 promoter
region
34
3.4 Cloning the intron into the enhancer region of pGL3 Basic and
pGL3N-1273
35
3.5 Transient transfections of NAP1L1 promoter deletion constructs
revealed regions between -442 bp and -297 bp that may contain a
positive regulatory element and -297 bp and -183 bp that may contain a
negative regulatory element
36
3.6 Preliminary transient transfection results from the smaller NAP1L1
promoter deletions between -442 bp and -297 bp as well as between
-297 bp and -183 bp relative to the TSS
39
3.7 Transient transfections of the NAP1L1 promoter and intron regions
41
3.8 Transient transfections of NAP1L1 promoter and intron regions with the
mutated c-Myc consensus sequence
42
Chapter 4: Discussion
45
4.1 Transient transfections of initial deletion constructs revealed a
regulatory element located within the region between -442 bp and
-297 bp relative to the transcription start site (+1) and smaller deletions
of this region suggest that the element lies between -367 bp and -335 bp
relative to the TSS
45
4.2 Transient transfections of deletion constructs revealed a regulatory
element located within the region between -297 bp and -183 bp relative
to the transcription start site (+1) and smaller deletions of this region
suggest that there is an element that lies between -297 bp and -254 and a
second element that lies between -254 bp and -215 bp relative to the
TSS
46
4.3 Transient transfections of initial deletion constructs revealed a potential
element between -183 bp and -56 bp relative to the TSS
48
4.4 Transfections of intron constructs revealed a functional c-Myc binding
site in the first intron of NAP1L1
48
4.5 Future directions
53
References
55
vi ABSTRACT
Promoter Characterization of the Mantle Cell Lymphoma Associated Gene, NAP1L1
By
Loni Hands
Master of Science in Biology
The precise regulation of gene expression within a cell determines the structure
and function of a given cell type. Dysregulation of normal gene expression may lead to
diseases such as cancer. Differences in gene expression among cancers of similar origin
may explain the differing phenotypes between more aggressive cancers and less
aggressive cancers. Mantle cell lymphoma (MCL), an aggressive cancer, expresses a
higher level of the nucleosome assembly protein 1-like 1 (NAP1L1) than small
lymphocytic lymphoma (SLL), a less aggressive cancer of similar origin. NAP1L1 is
involved in cell cycle and gene regulation, making it a candidate for involvement in
cancer progression.
In order to understand the mechanisms behind NAP1L1 expression, the putative
regulatory region of NAP1L1 from -1273 bp to +387 bp relative to the transcription start
site (TSS, +1) was cloned into the luciferase reporter vector pGL3 Basic. After transient
vii transfections of this construct into human embryonic kidney (HEK293T) cells confirmed
the ability of this region to regulate gene expression, deletions of the promoter region
were made and revealed the location of a positive regulatory element between -442 bp
and -297 bp and a negative regulatory element between -297 bp and -183 bp. Preliminary
data from transfections of smaller deletion constructs within each of these regions
narrowed down the location of the positive regulatory element to between -367 bp and 335 bp and potentially two negative regulatory elements between -297 bp and -254 bp
and between -254 bp and -215 bp. Further research will be necessary to determine the
exact identities of these elements.
In order to determine whether there is an enhancer element within the first intron
of NAP1L1, as there appears to be in mice (Wu et al., 2008), a region from +382 bp to
+1915 bp containing the beginning of the first intron was cloned into the pGL3 construct
containing the full isolated promoter region of NAP1L1 (-1273 bp to +387 bp). Transient
transfections of this construct with the intron in the “forward” direction resulted in
increased luciferase activity over 2-fold compared to the construct containing only the
full NAP1L1 promoter region. Interestingly, and unexpectedly, the construct containing
the full promoter region of NAP1L1 and the intron region in the reverse direction resulted
in luciferase activity that was comparable to the construct with the full promoter and no
intron. When the transcription factor binding sequence for c-Myc within the intron region
was mutated in the construct containing the full promoter region and intron in the forward
direction, the results were also similar to those of the construct containing only the full
promoter, suggesting a potential role for c-Myc in regulating NAP1L1 in a locationindependent, orientation-dependent manner.
viii CHAPTER 1: INTRODUCTION
1.1 Importance of gene regulation
Proper gene regulation is essential to the normal functions of the cell.
Coordinating the development of an organism, ensuring that genes are up regulated or
down regulated at the right time during each stage is the responsibility of gene regulatory
processes. Essentially, gene regulatory programs maintain the normal function of a given
cell type. Characteristics like cell shape and cell type-specific functions such as
immunoglobulin production and secretion by B lymphocytes are determined by the
regulated gene expression of that cell type. For example, in the case of immune cells,
differences in cell surface receptors and markers between cell types and cell signaling
pathways that lead to regulatory responses are important to an appropriate immune
system response to an infection. When this regulation is disrupted or prevented in any
way, diseases such as cancer or abnormalities in development can result.
1.2 Consequences of gene dysregulation
Dysregulation of certain genes or classes of genes can lead to diseases or
problems in development. Cancer, as an example, is a disease that results from the
dysregulation of apoptotic and cell cycle genes such as proto-oncogenes. Protooncogenes are drivers of the cell cycle; they are directly involved in pushing the cell
cycle forward (Vermeulen et al., 2003). When mutated or dysregulated, these oncogenes
will cause a cell to continue inappropriately dividing, eventually resulting in the
formation of a tumor (Vermeulen et al., 2003). One such example is the oncogene MYC,
1 which encodes the transcription factor c-Myc, which regulates genes involved in moving
the cell cycle forward and whose overexpression has been correlated with multiple
cancers (Tu et al., 2015; Wu et al., 2008). This transcription factor binds an enhancer box
along with the regulator Max to increase expression of cell cycle genes, such as cyclin
D1, and drive proliferation (Tu et al., 2015). Uncontrolled cell divisions will result in an
accumulation of mutations that will allow for tumor formation and even for the cancer to
become metastatic.
1.3 Gene regulation – Overview
In order to understand why the dysregulation of certain genes leads to diseases
such as tumor formation, the normal genetic regulation within various cell types must be
studied. The genetic composition within a cell is generally the same between cell types of
an organism, but it is the differences in genetic regulation that distinguishes one cell type
from another. This differential regulation maintains cell structure, shape, and
functionality of a given cell type. An epithelial cell may express some genes at higher or
lower levels than a B lymphocyte, or genes may be completely turned on or off.
Determining whether a vertebrate limb forms and with what polarity it forms is
determined by specific gene expression in a dose-dependent and even a locationdependent manner (Gehrke and Shubin, 2016). Mutations involved in diseases such as
cancer lead to the dysregulation of many genes that compromise the normal functions of
a cell.
Genes are regulated through the recognition of various DNA sequences by
regulatory proteins that collectively recruit RNA polymerase II (RNAPII) to transcribe a
2 gene or inhibit RNAPII from transcribing the gene. These DNA sequences are grouped
into categories termed promoters, enhancers, and silencers. Promoters are composed of a
core promoter region spanning from approximately -40 to +40 base pairs (bp) relative to
the transcription start site (TSS), denoted +1, and promoter proximal elements located a
few hundred base pairs upstream, and sometimes downstream, of the transcription start
site.
1.4 Gene regulation – Core promoters and transcription initiation
Core promoters contain short sequences to which basal transcription factors bind.
The basal transcription factor machinery is proving to be more complex than originally
thought (Danino et al., 2015). Previously, the basal TFs were thought to be similar
between genes and cell types, but more recent work has shown that different
combinations of subunits that can be cell type-specific (Danino et al., 2015, Goodrich and
Tjian, 2010). The most studied basal factors include TFIID, which usually contains the
TATA-binding protein (TBP) and 13-14 TBP-associated factors (TAFs), TFIIA, TFIIB,
TFIIF, TFIIE, and TFIIH (Goodrich and Tjian, 2010). Subunits within the TFIID
complex recognize and bind core promoter elements and then other basal factors
associate with TFIID (Goodrich and Tjian, 2010; Maston et al., 2006). The combination
of various basal factors and the mediator with RNAPII form the preinitiation complex
(PIC) (Dikstein, 2011; Goodrich and Tjian, 2010). The process of transcription initiation
differs between genes, but results when a combination of basal TFs and specific TFs
assemble and interact to recruit RNAPII to the promoter (Danino et al., 2015; Dikstein,
2011; Goodrich and Tjian, 2010). Depending on the type of promoter, the pattern of
3 transcription initiation will be different (Danino et al., 2015). There are many ways that
promoters have been classified, but two of the main classifications are focused versus
dispersed promoters (Danino et al., 2015). Focused promoters are more often cell typespecific and are generally characterized by a single transcription start site with core
elements, often with a TATA-box, positioned at specific distances from the TSS (Haberle
and Lenhard, 2016; Danino et al., 2015). Focused promoters are less common in
mammals, but are more highly conserved (Haberle and Lenhard, 2016). Dispersed
promoters, on the other hand, are more characteristic of ubiquitously expressed or
housekeeping genes and use multiple TSSs across a range of ~100 bp and are more likely
to contain CpG islands (Haberle and Lenhard, 2016). Dispersed promoters are the more
common type of promoter in humans (Haberle and Lenhard, 2016). Another
classification of promoters is into three classes: Type I, II, and III (Haberle and Lenhard,
2016). These types incorporate epigenetic information as well. Type I promoters are
somewhat similar to focused promoters, but include a less organized nucleosome
structure around the promoter region. Type II promoters are similar to dispersed
promoters and have specific locations of histones on either side of the. Type III
promoters are somewhat more dispersed than type II and are more characterized by
chromatin structural differences. Talk of a fourth promoter type for genes involved in
translation has come up but is less well studied (Haberle and Lenhard, 2016). Still, the
majority of differential regulation between cell types comes from the proximal promoter
elements, enhancers, and silencers involved in gene expression.
4 1.5 Gene regulation – Proximal promoter elements (PPEs)
Proximal promoter elements are composed of short sequences to which specific
transcription factors bind to increase or decrease transcription (Maston et al., 2006).
These groups of proximal promoter elements and corresponding transcription factors not
only differ between genes but the specific transcription factors expressed in one cell type
may not be expressed or may be expressed at a different level in a different cell type,
leading to variable expression of genes containing those elements. The general
mechanism by which these transcription factors bind and regulate expression through the
proximal promoter elements has to do with the transcription factor structure, the
interaction between these specific TFs, and the interactions between the specific TFs and
the basal factors that bind the core promoter (Maston et al., 2006). The common model
for the structure of the specific transcription factors is the presence of a DNA-binding
domain and an activating domain. The DNA-binding domains vary in structure and the
TFs are generally categorized based on the way they recognize and bind DNA sequences
(Maston et al., 2006). Often, the specific transcription factors bind DNA as homodimers,
interacting with a second copy of the TF, or heterodimers, interacting with a different
transcription factor, to regulate transcription (Maston et al., 2006). Many transcription
factors will interact with coactivators, which do not directly bind DNA but interact with
other proteins bound to DNA in the promoter region, in order to assist in regulating
transcription (Maston et al., 2006). These specific transcription factors and their
interactions with each other, with coactivators, and with the basal transcription machinery
display synergy with regards to their effects on transcription levels (Maston et al., 2006).
Although specific proximal promoter elements may have different functions in gene
5 expression, the main functions of PPEs include assisting in PIC assembly at the core
promoter, contributing to transcription initiation, core promoter escape, or re-initiation,
and recruiting chromatin remodeling complexes to affect the rate of transcription (Maston
et al., 2006). The core promoter and proximal promoter elements are location-dependent,
so removal or relocation of this region will significantly decrease or eliminate the
expression of the corresponding gene.
1.6 Gene regulation – Enhancers and silencers
Enhancer and silencer sequences are also bound by specific transcription factors,
but these sequences are location-independent, meaning they can be located relatively far
from the genes they regulate and that moving these regulatory sequences will generally
not affect gene expression (Maston et al., 2006). Enhancers are bound by activating
transcription factors, such as c-Myc, which increase gene expression, and silencers are
bound by repressors, transcription factors that decrease gene expression (Maston et al.,
2006). Enhancers, bound by specific transcription factors similar to or the same as those
that bind PPEs, will loop around to interact with the promoter of a gene, forming an
enhanceosome that increases transcription (Maston et al., 2006). This increase in activity
seems to be the result of the enhancer complex having the ability to keep specific
transcription factors and the basal factor complexes within the area of the promoter so
that re-initiation of transcription occurs more frequently (Engel et al., 2016). Gene
regulation also seems to be affected by the position of genes in lamina associated
domains (LADs) or topologically associated domains (TADs) within the nucleus. LADs
are near the periphery of the nucleus and are associated with genes that are silenced
6 (Engel et al., 2016). TADs are regions within the nucleus, whose boundaries are mostly
determined by CTCF and cohesin proteins, that form a unit where DNA is brought more
closely together to make it easier for distal enhancers to interact with the promoters they
regulate (Engel et al., 2016). The activity of enhancers is also influence epigenetically by
methylated histones (H3K27me3 and H3K4me1), correlating with decreased activity, and
acetylated histones (H3K27ac), correlating with increased activity (Engel et al., 2016).
Enhancers were previously characterized as an orientation-independent regulatory
element but there is evidence of enhancers behaving in an orientation-dependent manner
(Engel et al., 2016). For example, in different Drosophila species, a regulatory factor
called ABD-B contributes to sexually dimorphic coloring by binding to an enhancer
element that regulates the yellow gene (Jeong et al., 2006). In these different species, the
sequence of the element is slightly different, especially for more distantly related species
(Jeong et al., 2006). In one of the species that lacks this sexually dimorphic coloring, the
consensus sequence is reversed, or on the opposite strand, compared to species like D.
melanogaster which contain the normal consensus sequence on the forward strand of the
gene (Jeong et al., 2006). A gel shift assay revealed that the transcription factor still
bound to both sequences but no longer regulated the yellow gene in the species lacking
the distinguishing coloring (Jeong et al., 2006). In a second example, an enhancer located
between two divergent promoters loses its ability to upregulate one of the genes when
inverted in vitro (Swamynathan and Piatigorsky, 2002).
7 1.7 Gene expression in mantle cell lymphoma vs. small lymphocytic lymphoma
Identifying which of these regulatory elements are involved in the expression of a
particular gene between normal cells and abnormal cells (cancerous or of another disease)
of the same cell origin can help us understand the differential expression that may be
causative of certain diseases as well as differentiate between diseases, such as a more
aggressive cancer and a less aggressive cancer of similar cell origin.
As previously mentioned, dysregulation of cell cycle or apoptotic genes within a
cell can lead to cancer. Changes in expression of the regulatory factors that bind
promoters, as well as the chromosomal mutations that result in the movement of
regulatory elements, may lead to increases or decreases in gene expression. For example,
mantle cell lymphoma, an aggressive B cell cancer, has a characteristic translocation that
moves a regulatory element near the cyclin D1 gene, increasing cyclin D1 expression
(Henson et al., 2011). By understanding the specific causes of dysregulation that lead to a
disease such as cancer, possible targets for treatment can be identified and tested for
efficacy. Two such cancers that display differential gene regulation are mantle cell
lymphoma (MCL) and small lymphocytic lymphoma (SLL). Both are derived from
similar B-lymphocyte origins, but MCL has a more aggressive phenotype and a patient
survival time around 3-4 years, while SLL is less aggressive and has closer to a 10-year
patient survival time (Henson et al., 2011). The differential gene regulation between
these two lymphomas results in the phenotypic differences between the more aggressive
MCL and the less aggressive SLL, and so studying these differences may determine why
MCL is more aggressive than SLL and lead to the identification of potential targets for
treatment. Using a suppression subtractive hybridization (SSH) method, Henson and
8 colleagues identified the differential gene expression in MCL versus SLL (Henson et al.,
2011). One of the genes expressed differently between these two cancers is the
nucleosome assembly protein 1-like 1 (NAP1L1), which has the potential to be, in part,
causative of a cancerous phenotype.
1.8 Nucleosome assembly protein 1-like 1 (NAP1L1)
Nucleosome assembly proteins are a class of proteins involved in nucleosome
assembly, histone chaperoning, and cell cycle and gene regulation (Attia et al., 2011;
Okuwaki et al., 2010; Park and Luger, 2006). Nucleosome assembly protein 1 was first
discovered in Xenopus laevis and there have since been found five nucleosome assembly
protein 1-like proteins in humans, NAP1L1 through NAP1L5. Two of these NAP1-like
proteins are expressed ubiquitously (NAP1L1 and NAP1L4) and the remaining three are
expressed solely in neurons (Attia et al., 2011). NAP1L1 has been shown to have an
involvement in cell cycle progression and gene regulation (Schimmack et al., 2014).
Knockdown of NAP1L1 in BON cells (a human pancreatic neuroendocrine neoplasm cell
line) before transplantation into mice resulted in smaller tumors as well as no visible
signs of metastasis compared to the transplanted control BON cells, which showed larger
tumors and metastasis to the liver and peritoneum, suggesting a role for NAP1L1 in cell
proliferation (Schimmack et al., 2014). NAP1L1 involvement in gene regulation can be
seen in the results of a ChIP analysis of NAP1L1 binding the promoter of the tumor
suppressor p57Kip2 and a correlation between decreased methylation patterns at the
p57Kip2 promoter and the absence of NAP1L1 (Schimmack et al., 2014). In mice,
NAP1L1 appears to be regulated by c-Myc, as a ChIP-seq of MYC brings down the
9 promoter region (approximately -2000 bp to +2000 bp) of NAP1L1 and two c-Myc
consensus sequences are found within the first intron (Wu et al., 2014). There also
appears to be a strong correlation between high expression of NAP1L1 and high
expression of MYC in human B cell lymphomas, suggesting a potential interaction or
regulatory relationship between these two genes in humans as well (Wu et al., 2014).
NAP1L1 is also involved in histone chaperoning and has a higher nucleosome
disassembly activity than the other ubiquitously expressed nucleosome assembly protein
NAP1L4 (Okuwaki et al., 2010). NAP1L1 overexpression is correlated with many
cancers such as pancreatic neuroendocrine neoplasms, small intestinal carcinoids, and
mantle cell lymphoma (Kidd et al., 2006; Schimmack et al., 2014; Henson et al., 2011).
1.9 Characterizing the NAP1L1 promoter region and enhancer element
Collectively, these findings suggest that human NAP1L1 may have a causative
role in the aggressive nature of mantle cell lymphoma. Evidence of MYC binding the
mouse NAP1L1 promoter region suggests that the human homolog may be regulated by
the oncogene MYC through a conserved enhancer element within the first intron of
NAP1L1 (Wu et al., 2008). NAP1L1 also appears to have a role in regulating cell cycle
genes and so when overexpressed this protein may affect cell cycle or apoptotic genes
and lead to an increase in proliferation. Due to its role in cell proliferation and gene
regulation, it is possible that overexpression of this gene may be causative of the
aggressive nature of mantle cell lymphoma. If NAP1L1 is found to be causative of an
aggressive phenotype when overexpressed, then it may be a potential target for treatment.
Identifying which DNA elements and transcription factors are involved in NAP1L1
10 expression will provide insight as to the methods involved in the normal regulation of this
gene as well as what factors may contribute to the dysregulation of this gene. The goal of
this project is to characterize the promoter region and identify a potential enhancer
element within the first intron of NAP1L1 in order to elucidate in part the method of
regulation of NAP1L1.
11 CHAPTER 2: METHODS
2.1 Cloning the NAP1L1 promoter region into pGL3 Basic. A region of 1431 base
pairs (bp), spanning from -563 bp to +868 bp relative to the transcription start site (+1),
was amplified from human genomic DNA by PCR using the forward primer 5’–AGT
CAA TTC ATC ATC ACT TG–3’ and reverse primer 5’–TCC GCC GTT CTT TTC
ATC TC–3’. The thermocycler was set for an initial denaturation of 95˚C for 5 min, 35
cycles at 95˚C for 30 s, 46˚C for 60 s, and 72˚C for 90 s, and a final extension of 72˚C for
5 min. The largest two of three fragments were gel extracted and purified using
QIAGEN’s Gel Extraction Kit and restriction digested with PstI to identify the correct
fragment. The 5’ ends of the correct fragment were phosphorylated using the protocol
and reagents from Lucigen’s pGC Blue cloning kit and subsequently purified using
QIAGEN’s PCR Purification Kit. The purified, phosphorylated fragment was then ligated
into pGC Blue using the buffer and T4 DNA ligase from Lucigen’s pGC Blue cloning kit
and protocol. To the ligation reaction, 5 µL of this purified fragment (~13.2 ng/µL), 2.5
µL of the 4X pGC Blue Vector Premix, 1 µL of CloneSmart DNA Ligase (2 U/µL), and
autoclaved D.I. water to fill to 10 µL were added, mixed, and allowed to incubate at room
temperature for 2 h. After 2 h, the ligation reaction was heat denatured at 70˚C for 15 min
and transformed into Zymo Research’s Mix & Go Competent Cells following the
manufacturer’s protocol. The cells were transformed with 5 µL of the ligation reaction
and, before plating, 4 volumes of plain LB was added to the transformation mixture and
the cells were allowed to recover for 1 h at 37˚C in a shaking incubator. From this
mixture, 100 µL was plated onto one prewarmed (37˚C) LB-kanamycin (50 µg/mL) plate
12 and 10 µL was plated onto a second prewarmed LB-kanamycin plate. The following day,
the remaining 400 µL was plated onto two prewarmed LB-kanamycin plates (200 µL per
plate). Seven colonies were placed in liquid LB broth containing kanamycin and left
overnight at 37˚C in a shaking incubator. The following morning these liquid cultures
were boil prepped (See boiling lysis plasmid purification protocol below). Each plasmid
prep was restriction digested with EcoRI to determine if the plasmid contained an insert
and with SacI to determine the orientation of the insert. The plasmid containing the
promoter region oriented towards the HindIII restriction site in pGC Blue was used for
cloning into the luciferase reporter vector pGL3 Basic (Promega). A sequential digest
first with HindIII and then with XhoI removed the promoter from pGC Blue and inserted
into pGL3 Basic (do I need to talk about gel extraction here?) (See sequential restriction
digest details under the restriction digest protocol below). Promega’s 10X Ligase Buffer
(300 mM Tris-HCl pH 7.8 at 25˚C, 100 mM MgCl2, 100 mM DTT, and 10 mM ATP)
and T4 DNA Ligase (3 U/µL) were used to set up a 10 µL ligation reaction with 5 µL of
purified insert (~15.2 ng/µL) and 3 µL of purified pGL3 Basic (~25 ng/µL). Following
the protocol provided by Zymo Research, 5 µL of the ligation mixture was transformed
into Mix & Go Competent Cells. These cells were plated directly onto prewarmed (37˚C)
LB-ampicillin plates. One LB-ampicilling plate received 10 µL of the transformation
mixture and another received 95 µL. All 17 colonies from the transformation were placed
in 3 mL liquid LB medium containing ampicillin and grown overnight. These overnight
cultures were boil prepped and the uncut plasmids were run on a gel next to a sample of
uncut pGL3 Basic. Five plasmid preps were then digested with EcoRI to determine the
presence of an insert and XbaI to determine the orientation of the insert. Since the reverse
13 primer annealed within the first intron of NAP1L1, the intron needed to be removed from
this recombined plasmid. A restriction digest with SpeI removed the intron and the
plasmid was ligated onto itself to complete the removal of the intron. The confirmed
recombined plasmid was then purified with QIAGEN’s Spin Miniprep Kit and sent for
sequencing by Laragen.
2.2 Cloning a larger fragment of the NAP1L1 promoter region into pGL3 Basic. The
larger fragment, 1945 bp, extends from -1273 bp to +672 bp relative to the transcription
start site (+1). An amplified region of the NAP1L1 promoter was obtained through PCR
using the forward primer 5’–AGA AGG GTG TAC AGG AAT AGG C–3’ and reverse
primer 5’–GGT GGA GCT CAG AGA ACC TTA G–3’. A primer kinase reaction was
set up for the primers following the pGC Blue Cloning Kit protocol (Lucigen). A PCR
reaction was then set up using 1 µL of this primer kinase reaction, 50 ng genomic DNA
and Promega’s GoTaq Green Master Mix in a 50 µL reaction. A touchdown PCR
reaction was programmed into the thermal cycler so that there was an initial denaturation
step at 95˚C for 5 min, 2 cycles of 95˚C for 30s, 61˚C for 40 s, and 72˚C for 2 min, 2
cycles of 95˚C for 30s, 58˚C for 40 s, and 72˚C for 2 min, 2 cycles of 95˚C for 30s, 55˚C
for 40 s, and 72˚C for 2 min, 2 cycles of 95˚C for 30s, 53˚C for 40 s, and 72˚C for 2 min,
27 cycles of 95˚C for 30s, 51˚C for 40 s, and 72˚C for 2 min, a final extension of 72˚C for
5 min, and a hold at 4˚C. The reaction was run on a gel and the band was extracted and
purified using QIAGEN’s Gel Extraction Kit. Using the protocol in the pGC Blue
Cloning Kit, the phosphorylated NAP1L1 promoter region was ligated into pGC Blue
using 6.5 µL of the gel extracted promoter (~17.3 ng/µL) and 4 µL of this ligation was
14 transformed into Lucigen’s chemically competent E. Cloni cells with an outgrowth step
that includes the addition of 260 µL of Recovery Medium (Lucigen) and incubation at
37˚C (250 RPM) for 1 h before plating onto prewarmed LB plates containing kanamycin
(50 µg/ml). Overnight cultures were set up from resulting colonies and purified by
boiling lysis. Purified plasmids were digested with EcoRI and SpeI to confirm identity
and orientation of insert. In order to clone directionally into pGL3 Basic, pGL3 Basic and
a pGC Blue plasmid containing the NAP1L1 promoter region pointing towards the
HindIII site in pGC Blue was digested first with HindIII and then with XhoI, increasing
the salt concentration from 50 mM to 150 mM in between (see sequential digest
protocol). Both the digested pGL3 Basic and NAP1L1 promoter region were gel extracted
and purified using QIAGEN’s Gel Extraction Kit. A ligation was then set up using 7 µL
of pGL3 Basic (~3.9 ng/µL), 1 µL NAP1L1 (~36.1 ng/µL), 1 µL 10X Ligase Buffer
(Promega), and 1 µL T4 DNA Ligase (3 U/µL) (Promega) for a total volume of 10µL
that was incubated at room temperature for 3 h. The ligation was inactivated at ~74˚C for
15 min and 4 µL was transformed into Lucigen’s chemically competent E. Cloni cells
following their protocol with the outgrowth step. Overnight cultures of resulting colonies
were purified by boiling lysis and confirmed for identity and orientation of insert using
EcoRI and NotI. The confirmed recombined plasmid was then digested with SpeI and
ligated onto itself to remove the intron that was initially amplified with the promoter
region, leaving the remaining fragment that spans from -1273 bp to +387 bp relative to
the transcription start site. The final pGL3N-1273 was then purified using Thermo
Scientific’s GeneJET Plasmid Miniprep Kit for transfection.
15 2.3 Cloning a region of NAP1L1’s intron 1 into pGL3N-1273 and pGL3 Basic. A
1913 bp region containing part of the first intron of NAP1L1, including the putative myc
binding site, was amplified by PCR using the forward primer 5’–AAG ATA TGG TGG
GGT GCT TAA C–3’ and reverse primer 5’–TCT AAA ATA CGG GCT CCT TGA G–
3’. The primers were phosphorylated using the primer kinase reagents and protocol from
Lucigen’s pGC Blue Cloning Kit. A 50 µL PCR reaction was set up using 50 ng genomic
DNA, 1 µL primer kinase reaction, and Promega’s 2X GoTaq Green Master Mix. A
touchdown PCR was programmed into the thermal cycler so that there was an initial
denaturation at 95˚C for 5 min, 2 cycles of 95˚C for 30 s, 59˚C for 40 s, and 72˚C for 2
min, 2 cycles of 95˚C for 30 s, 56˚C for 40 s, and 72˚C for 2 min, 2 cycles of 95˚C for 30
s, 53˚C for 40 s, and 72˚C for 2 min, 2 cycles of 95˚C for 30 s, 51˚C for 40 s, and 72˚C
for 2 min, 27 cycles of 95˚C for 30 s, 49˚C for 40 s, and 72˚C for 2 min, a final extension
of 72˚C for 5 min, and a hold at 4˚C. The resulting band was gel extracted and purified
using QIAGEN’s Gel Extraction Kit. This purified fragment was then ligated into pGC
Blue following Lucigen’s protocol, adding 6.5 µL of the phosphorylated intron fragment
(~26.1 ng/µL) to the reaction. The ligation was inactivated at 74˚C for 15 min and 4 µL
of the ligation was transformed into Lucigen’s chemically competent E. Cloni cells
including an outgrowth step and the entire mixture was plated onto prewarmed LB plates
containing kanamycin. Overnight cultures were set up from resulting colonies and the
plasmids were purified by boiling lysis. Plasmid identities and insert orientations were
confirmed by digesting with EcoRI and SpeI. In order to clone into pGL3N-1273 and
pGL3 Basic, the two vectors were cut with SalI, blunted, CIP-ed, and gel extracted and
purified using QIAGEN’s Gel Extraction Kit. The pGC Blue vector with the intron facing
16 the SpeI site in pGC Blue was cut with SpeI to remove it from pGC Blue and at the same
time remove the majority of the 5’–UTR region that was part of the original amplified
fragment. This fragment, now 1534 bp, was also blunted and gel extracted and purified
using QIAGEN’s Gel Extraction Kit. The intron was then ligated into pGL3 Basic and
pGL3N-1273 using ~40 ng of intron at between 24 and 30 ng of pGL3 Basic and between
18 and 24 ng of pGL3N-1273. These ligations were transformed into chemically
competent E. coli. Overnight cultures of resulting colonies were purified by boiling lysis
and digested with EcoRI and SacI to confirm identity and intron orientation. Confirmed
constructs were miniprepped using Thermo Scientific’s GeneJET Plasmid Miniprep Kit
and a second confirmation was done by digesting the plasmids with BglI, SalI & XbaI,
and SacI.
2.4 Boiling lysis plasmid purification. A master mix of boil buffer (0.5% Triton X-100,
8% sucrose, 50 mM EDTA, 10 mM Tris, pH 8) and lysozyme (10 mg/mL) was made in a
13:1 ratio, respectively. The master mix was most often made for the number of samples
plus one, with each sample requiring 350 µL of master mix (325 µL boil buffer and 25
µL lysozyme). Overnight cultures were decanted into 1.5 mL microcentrifuge tubes and
centrifuged at maximum speed (13,000 RPM) for 15 s, waiting until the centrifuge
reached maximum speed and then counting 15 s. The supernatant was decanted and
another 1.5 mL of culture was added and centrifuged at max speed for 15 s. The
supernatant was decanted and tubes were inverted on paper towels to drain as much of
the liquid supernatant as possible. To each bacterial pellet, 350 µL of the master mix was
added and each tube was dragged across a peg rack to resuspend the pellet. The tubes
17 were placed in a boiling water bath for 40 to 45 s. Once the tubes were removed, they
were allowed to cool to approximately room temperature and then centrifuged at max
speed for 5 min. The gooey cell debris containing genomic DNA in each tube was
removed carefully with a toothpick. To each tube, 40 µL of 3 M NaOAc was added, the
tube was vortexed briefly, 425 µL of isopropanol was added, the tube was vortexed
briefly, and the tube was centrifuged at max speed for 5 min. The supernatant was
decanted and tubes were inverted to air dry before they were resuspended in 100 µL of
RNase water (10 µg/mL).
2.5 Restriction digests. Restriction digests were done according to either Promega’s or
Thermo Fisher’s protocols. For the sequential digests necessary for cloning the original
NAP1L1 promoter region (both the -563 and -1273 promoter regions) there was an
adjustment of salt concentration between digests. The first digest with HindIII was done
as Promega’s protocol recommends with 10X Buffer B (1X concentration: pH 7.5 @
37˚C, 6 mM Tris-HCl, 6 mM MgCl2, 50 mM NaCl, 1 mM DTT). After heat-inactivating
HindIII, the digest volume was brought up to a final volume 40 µL with the addition of
3.33 µL 10X Buffer D (1X concentration: pH 7.9 @ 37˚C, 6 mM Tris-HCl, 6 mM
MgCl2, 150 mM NaCl, 1 mM DTT) in order to increase the salt concentration from 50
mM to 150 mM for the XhoI digest. The digests were then run on a gel and desired bands
were cut out and purified.
2.6 Transcription factor consensus sequence identification. Potential transcription
factor binding sites within the promoter region were identified by means of similarity
18 between the isolated NAP1L1 promoter region sequence (-1273 bp to +387 bp, relative to
the transcription start site) and transcription factor consensus sequences using the
following bioinformatics software: Match, Alibaba 2, and PROMO. The parameters in
Match were set so “group of matrices” was set to “vertebrates”, “cut-off selection for
matrix group” to “to minimize the sum of both error rates”, and the “predefined profiles”
to “best_selection.prf” (http://www.gene-regulation.com/cgibin/pub/programs/match/bin/match.cgi). The parameters for Alibab2 were unchanged
from the default and included “pairism to known sites” set to “50”, “mat. width in bp” set
to “10”, “min number of sites” set to “4”, “min mat. conservation” set to “75%”, “sim of
seq to mat.” set to “1%”, and “factor class level” set to “4 (e.g. RAR-b’)”
(http://www.gene-regulation.com/pub/programs/alibaba2/index.html). The parameters for
ALGGEN were set so that under “SelectSpecies” the “factor’s species” and “site’s
species” were both set to “human, Homo sapiens” and under “SearchSites” the
“maximum matrix dissimilarity rate” was set to “5” (http://alggen.lsi.upc.es/cgibin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). After a list of putative transcription
factor binding sites (TFBSs) was obtained from each program, the lists were compared
and TFBSs similar between at least two of the programs were noted and organized into
Figure 1.
2.7 Designing initial deletion constructs of the NAP1L1 promoter region. Using the
results of the transcription factor bioinformatics programs, deletion constructs were
designed to remove segments that each increased in size by approximately 200 base pairs
and attempt to avoid cutting directly in the middle of potential transcription factor
19 bindings sites. Using site-directed mutageness (QuikChange Site-Directed Mutagenesis
Kit, Agilent), single nucleotide changes were made to introduce restriction sites within
the promoter regions at locations -442 bp, -297 bp, -183 bp, and -56 bp relative to the
transcription start site (+1). The primers containing the mutations (bolded) for each of the
deletion constructs can be found in Table 1. The primers for constructs pGL3N-442,
pGL3N-297, and pGL3N-183 were designed by centering the mutation within the primer
and encompassing a region that extended ~15 nucleotides on either side of the mutation.
The primers were designed by hand except for the primers for pGL3N-56, which were
designed by Agilent’s free online QuikChange Primer Design program
(http://www.genomics.agilent.com/primerDesignProgram.jsp). Using the protocol and
reagents provided by Agilent’s QuikChange Site-Directed Mutagenesis Kit, the first set
of deletion constructs (pGL3N-442, -297, -183, and -56) were made. The PCR protocol
and DpnI digestion were followed exactly as depicted in the protocol. The PCR products
were then transformed into chemically competent E. coli DH5-a. Resulting colonies were
set up in overnight liquid cultures (LB-Amp) and boil prepped the following day. The
purified plasmids were digested using the corresponding restriction enzyme designed to
take out a fragment of the promoter. After confirming the removal of a piece of the
promoter from each construct on a gel, the larger fragments were gel extracted using
QIAGEN’s Gel Extraction Kit. The fragments were ligated on themselves using
Promega’s 10X Ligase buffer and T4 DNA Ligase (3 U/µL) in a 10 µL reaction. The
ligation mixtures were transformed into chemically competent E. coli DH5-a. The
resulting colonies were set up in overnight cultures and boil prepped the following day.
These purified plasmids were digested with KpnI and XbaI to confirm their identities.
20 The plasmids were repurified (from the corresponding colonies) using QIAGEN’s Spin
Miniprep Kit and sent out for sequencing at Laragen.
2.8 Designing smaller deletion constructs of the NAP1L1 promoter region. All
primers for site-directed mutagenesis can be found in Table 1 and were designed by hand
except for pGL3N-335, which was designed by Agilent’s free online QuikChange Primer
Design program (http://www.genomics.agilent.com/primerDesignProgram.jsp). The
second set of deletion constructs was made using a similar method to the first set. The
constructs pGL3N-398, pGL3N-335, and pGL3N-215 were made following the
QuikChange protocol with a few small adjustments. The PCR reactions contained a lower
annealing temperature of 45˚C and the extension time was increased to 7 min. The
amount of template (pGL3N-1273) that was used was 25 ng and the remainder of the
PCR protocol remained the same. The PCRs were digested with DpnI and 5 µL of each
PCR was transformed into Mix & Plate chemically competent E. coli cells (GeneChoice),
including an outgrowth with the addition 220 µL Recovery Medium (Lucigen) and
incubation at 37˚C (250 RPM) for 1 h before plating the entire mixture onto prewarmed
LB plates containing ampicillin. Overnight cultures of the resulting colonies were
purified by boiling lysis and the purified plasmids were double-digested with SmaI &
BglII (pGL3N-398), SmaI & NdeI (pGL3N-335), and SmaI & EcoRV (pGL3N-215). The
digests were run on a gel and the larger band was cut out and purified using QIAGEN’s
Gel Extraction Kit. The linearized plasmids pGL3N-398 and pGL3N-335 were cut with
BglII and NdeI, respectively, and so were blunted with Klenow and repurified with
QIAGEN’s PCR Purification Kit. The other construct, pGL3N-215, was cut with EcoRV
21 which leaves a blunt end and so did not require these additional steps. The linearized,
blunted plasmids were then ligated onto themselves using Promega’s 10X Ligase Buffer
and T4 DNA Ligase (3 U/µL). To each reaction 8 µL of plasmid was added (purified
plasmid concentrations: pGL3N-398 = ~5.1 ng/µL, pGL3N-335 = ~6.6 ng/µL, and
pGL3N-215 = ~3.9 ng/µL). The ligations were heat-inactivated before transforming 5 µL
of each into Mix & Plate chemically competent E. coli cells (GeneChoice), including an
outgrowth with the addition of 220 µL Recovery Medium (Lucigen) and incubation at
37˚C (250 RPM) for 1 h before plating the entire mixture onto prewarmed LB plates
containing ampicillin. Overnight cultures of resulting colonies were purified by boiling
lysis and all purified plasmids were digested with BglI and KpnI to confirm the deletion
of a region of the promoter. Confirmed deletion constructs were then purified using
Thermo Scientific’s GeneJET Plasmid Miniprep Kit.
For pGL3N-367 and pGL3N-254 a different proofreading polymerase was used, Phusion
DNA polymerase (Thermo Scientific), so some adjustments to the protocol were made.
The PCR reaction for pGL3N-254 contained 10 µL 5X Phusion HF Buffer, 50 ng
template DNA (pGL3N-1273), 125 ng of each primer (Table 1), 1 µL dNTP Mix (10 mM
each), and 0.5 µL Phusion DNA polymerase (2 U/µL). The thermocycler was set with an
initial denaturation step of 95˚C for 30 s, 18 cycles of 95˚C for 30 s, 45˚C for 1 min, 72˚C
for 6 min 40 s, and a hold at 4˚C. PCR reactions were then digested with DpnI for ~2 h,
EtOH precipitated, and resuspended in 4 µL Elution Buffer (Thermo Scientific). The
entire 4 µL was then transformed into Lucigen’s chemically competent E. Cloni cells.
Overnight cultures of the resulting colonies were purified through boiling lysis. The
purified plasmids were digested first with SmaI and then with BglII using Promega’s
22 buffers, increasing the salt concentration for the BglII digest using 10X Buffer D as
previously described (see sequential digest protocol). This digest was run on a gel and the
larger band was cut out of the gel and purified using QIAGEN’s Gel Extraction Kit. The
purified, linear fragment was blunted using Klenow as BglII produces overhangs.
Klenow was heat-inactivated and the blunted fragment was re-purified using QIAGEN’s
PCR Purification Kit. The vector was then ligated onto itself (8 µL at ~8.3 ng/µL) using
Promega’s 10X Ligase Buffer (1 µL) and T4 DNA Ligase (1 µL at 3 U/µL) for a total
reaction volume of 10 µL. The ligation reaction was heat-inactivated at ~70˚C for 15 min
and 5 µL was transformed into Mix & Plate chemically competent E. coli cells
(GeneChoice), including an outgrowth with 420 µL of Recovery Medium (Lucigen) and
incubation at 37˚C (250 RPM) for 1 h. The entire transformation mixture was then plated
onto prewarmed LB plates containing ampicillin. Overnight cultures of the resulting
colonies were purified by boiling lysis. The purified plasmids were digested with BglI
and KpnI separately to confirm the deletion of a fragment of the promoter region.
Confirmed constructs were then purified using Thermo Scientific’s GeneJET Plasmid
Miniprep Kit.
The construct pGL3N-367 was made similarly to pGL3N-254. The PCR reaction, DpnI
digestion, EtOH precipitation, transformation, and boiling lysis plasmid purification were
identical to the conditions for pGL3N-254 except for the primers used in the PCR
reaction (Table 1). This construct was then digested first with SmaI and then PvuII with
no increase in salt concentration as it was not required for PvuII. The digests were run on
a gel and the upper band was cut out and purified using QIAGEN’s Gel Extraction Kit.
As SmaI and PvuII are restriction enzymes that result in blunt-ended DNA strands, the
23 plasmid was directly ligated onto itself (6 µL) using Promega’s 10X Ligase Buffer (1 µL)
and T4 DNA Ligase (1µL at 3 U/µL) for a total reaction volume of 10 µL. The reaction
was incubated at room temperature for 3 h, inactivated at ~70˚C for 15 min, and 4 µL
was transformed into Mix & Plate chemically competent E. coli cells (GeneChoice),
including an outgrowth step with 420 µL Recovery Medium (Lucigen) and incubating at
37˚C (250 RPM) for 1 h) before the entire mixture was plated onto prewarmed LB plates
containing ampicillin. Overnight cultures of resulting colonies were purified by boiling
lysis and the purified plasmids were digested with BglI and KpnI separately to confirm
the deletion of a fragment of the promoter region. The confirmed plasmid was then
purified using Thermo Scientific’s GeneJET Plasmid Miniprep Kit.
2.9 Mutating the myc binding site within the first intron of NAP1L1. In order to
mutate the consensus sequence for the myc binding site withing the first intron of
NAP1L1 the QuikChange site-directed mutagenesis kit was used with some different
reagents and adjustments to the protocol, including some influence from the QuikChange
XL site-directed mutagenesis kit as the template exceeded 8000 bp. The primers used to
mutate the sequence can be found in Table 1. Thermo Fisher’s Phusion DNA polymerase
was used in this mutagenesis instead of the PfuTurbo DNA polymerase from the
QuikChange site-directed mutagenesis kit. Four 50 µL PCR reactions were set up,
containing 10 µL of 5X Phusion HF buffer, 50 ng of template (pGL3N-1273 + intron(F)),
125 ng of each primer (see Table 1), 1 µL of dNTP mis (10 mM each), 3 µL DMSO, and
1 U Phusion DNA polymerase. The thermocycler was set with an initial denaturation step
at 95˚C for 1 min, 18 cycles of 95˚C for 50 s, 45˚C for 50 s, and 72˚C (Phusion PCR
24 protocol specified 72˚C for an extension temperature) for 8 min 15 s, a final extension at
72˚C for 7 min, and a hold at 4˚C. These four reactions were first digested with DpnI at
37˚C for 2 h, then the four reactions were combined, EtOH precipitated, and resuspended
in 4 µL Elution Buffer (Thermo Scientific). This entire volume was transformed into
chemically competent E. coli with an outgrowth step including the addition of 260 µL of
recovery medium and incubation at 37˚C (250 RPM) for 1 h before plating the entire
mixture onto LB plates containing ampicillin. Overnight cultures were boiled prepped
and checked for correct plasmid size by digesting with BglI and ApaI. Plasmids of the
correct size were miniprepped using Thermo Scientific’s GeneJET Plasmid Miniprep Kit
and sent to Laragen for sequencing.
Table 1. Primers used for site-directed mutagenesis of promoter region and intron.
Construct
Promoter
Region
(relative to
TSS +1)
pGL3N-442
-442 to +387
pGL3N-398
-398 to +387
pGL3N-367
-367 to +387
Forward
Primer (5’-3’)
Reverse Primer
(5’-3’)
CCT CAT
TAT AAC
GCG TCC
AGA GAA
TGC AC
CCA TTT
TCA TCC
GTC AAA
AGA TCT
GAA GCC
GAA CTG
ACG
CTG ACG
GAC TGT
ACA TCA
GCT GCG
AGG ATG
TGG CG
GTG CAT
TCT CTG
GAC GCG
TTA TAA
TGA GG
CGT CAG
TTC GGC
TTC AGA
TCT TTT
GAC GGA
TGA AAA
TGG
CGC CAC
ATC CTC
GCA GCT
GAT GTA
CAG TCC
GTC AG
25 Engineered
Restriction Site
MluI
ACGCGT
BglII
AGATCT
PvuII
CAGCTG
pGL3N-335
pGL3N-297
pGL3N-254
-335 to +387
-297 to +387
-254 to +387
pGL3N-215
-215 to +387
pGL3N-183
-183 to +387
pGL3N-56
-56 to +387
(not relevant)
pGL3N-1273 +
myc binding
intron (F) –
site was
myc mut
mutated
GGG GTT
GGG GGG
CAT ATG
ATG ACG
AGC ACC C
GGC GCG
GAG ACC
CGC TAG
CAT AGG
AGA AGC
AAA AC
GAG GCC
GCC GAA
GAG ATC
TAG AGG
AAA AAA
CTG
CTT AGG
CGG GGA
AGG ATA
TCG GTT
ACC GGG
GGC CGG
CGA GTT G
CCG GCG
AGT TGC
AGG AAT
TCG GCC
TTG ACA
GCC GGG
CGC CGG
GCA CCC
GGG CCA
GGC CTT
CCC
GGG TGC
TCG TCA
TCA TAT
GCC CCC
CAA CCC C
CGG CGG
CTT CGC
TCC ATT
TGT GGT
AAT GGG
CTT CAT G
CAT GAA
GCC CAT
TAC CAC
AAA TGG
AGC GAA
GCC GCC G
26 NdeI
CATATG
GTT TTG CTT
CTC CTA
NheI
TGC TAG
GCTAGC
CGG GTC
TCC GCG CC
CAG TTT TTT
CCT CTA
BglII
GAT CTC
AGATCT
TTC GGC
GGC CTC
CAA CTC
GCC GGC
CCC CGG
TAA CCG
ATA TCC
TTC CCC
GCC TAA G
TGT CAA
GGC CGA
ATT CCT
GCA ACT
CGC CGG
GGG AAG
GCC TGG
CCC GGG
TGC CCG
GCG CCC
GGC
EcoRV
GATATC
EcoRI
GAATTC
SmaI
CCCGGG
Not a
restriction site.
Mutated the
consensus for
myc from
“CACGTG” to
“TTTGTG”
2.10 Transfection of promoter constructs into human embryonic kidney (HEK293T)
cells. The following protocol is slightly modified from QIAGEN’s Effectene Reagent
protocol for transient transfection of adherent cells. HEK293T cells were counted and
seeded in 12-well plates at a density of 2 x 105 cells per well with 1 mL of DMEM 10%
FBS medium. The number of plates needed was determined by the number of constructs
that were to be tested. Each construct was transfected into three separate wells so that the
luciferase assay results from the three wells could be averaged for each construct after
each transfection. After the cells were seeded, the 12-well plates were placed in the
incubater at 37˚C with 5% CO2 for approximately 28-30 h after seeding. The plasmid
control constructs, pRL-SV40, pGL3 Basic (no promoter), pGL3 Promoter, and pGL3
Control, were transfected alongside all of the NAP1L1 promoter and intron constructs.
All plasmids used in transfections were purified using Thermo Scientific’s GeneJET
Plasmid Miniprep Kit. Plasmid concentrations were determined using a
spectrophotometer. On the day of the transfection, master mixes were made and the
media on the cells was changed ahead of the transfection procedure. The media was
changed after cell confluency was determined. The existing media was removed, cells
were washed once with 1 mL of 1X PBS, 800 µL of media was added to each well, and
the cells were placed back into the incubator. Master mixes were prepared for the number
of wells receiving a pGL3 construct plus one. For a transfection in which one construct
was transfected into 3 wells, a master mix was made for 4 wells. The following portion of
the protocol describes the procedure for transfection of each pGL3 construct into 3 wells
and was scaled up if transfecting each construct into 4 wells. A master mix for each
pGL3 construct was prepared in a 2.0 mL microcentrifuge tube by first diluting each
27 pGL3 construct to 50 ng/µL in Thermo Scientific’s Elution Buffer at a total volume of 16
µL. Each well should receive 200 ng of the corresponding pGL3 construct. In a separate
microcentrifuge tube, the normalization construct pRL-SV40 was also diluted to 50
ng/µL in Elution Buffer (Thermo Scientific) to a volume large enough to accommodate
each well receiving 100 ng of pRL-SV40, so each master mix would receive 8µL. The
diluted pRL-SV40 was then added to each pGL3 master mix tube and the total volume
was diluted with Buffer EC (QIAGEN’s Effectene Reagent Kit) to a total volume of 300
µL. As the DNA to Enhancer (QIAGEN) ratio needed to remain constant, 9.6 µL
Enhancer was added to each master mix, the mixtures were vortexed for 1 s, and left to
incubate at room temperature for at least 5 min. The tubes were briefly centrifuged to
bring the liquid down to the bottom of the tubes and 4 µL of Effectene Reagent
(QIAGEN) was added. The tubes were vortexed for 10 s and incubated at room
temperature for at least 10 min. To each tube, 1600 µL warmed media was added and
mixed. From each master mix, 375 µL was added to each of 3 wells receiving a specific
construct. The plates were swirled and left to incubate at 37˚C with 5% CO2 for
approximately 48-56 h.
2.11 Harvesting transiently transfected HEK293T cells. Protocol modified from
Promega’s Dual-Luciferase Reporter Assay System manual. First, the media was
removed from each well and the cells were washed with 1 mL 1X PBS and swirled
gently. The PBS was then removed and 250 µL of 1X PLB (Passive Lysis Buffer,
Promega) was added. For each well, the cells were removed by pipetting up and down to
break up cell clumps and washing the bottom of the wells to make sure all of the cells
28 came off and were in the liquid. This lysate was then transferred to a microcentrifuge
tube and vortexed. All of the tubes, one for each well, were incubated in a dry-ice ethanol
bath for 5 min followed by a 5 min incubation in a 37˚C water bath. The tubes were then
placed back in the dry-ice ethanol bath and the incubation cycle was repeated two more
times, sometimes with the addition of vortexing between the 37˚C incubation and the dryice ethanol incubation. The lysates were then assayed for luciferase activity or stored in
the -20˚C or -80˚C until a later time for the luciferase assay.
Note: Would often stop after removing the lysates from the plates and vortexing the tubes
and continue with the dry-ice ethanol incubation and 37˚C incubation another day. Tubes
would be stored in the -20˚C freezer until that time.
2.12 Dual-Luciferase Assay of transfected pGL3 constructs. Luciferase assays were
carried out using Promega’s reagents from the Dual-Luciferase Reporter Assay System
and the Monolight 2010 luminometer. The luminometer was programmed to calculated
the normalization ratio of firefly luciferase activity (resulting from the pGL3 constructs)
to Renilla luciferase activity (resulting from pRL-SV40).
29 CHAPTER 3: RESULTS
In order to understand how the NAP1L1 gene is regulated, bioinformatics analyses
of the promoter region were used before the in vitro experiments. This data was used as a
guide in order to design deletion construct approximately every ~200 bp. If there was a
putative transcription factor binding site near one of the cut site locations, then the
deletion was designed around this cut site.
3.1 Bioinformatics analyses of the NAP1L1 promoter region.
Bioinformatics software was used to identify consensus sequences of transcription
factor binding sites (TFBSs) within the sequence of the NAP1L1 promoter region. The
bioinformatics programs Alibaba2, Match, and PROMO were used to identify these
consensus sequences. Each program generated a list of transcription factors and their
corresponding TFBSs within the region along with the location of each binding site. The
lists were compared and matching results from two or more of the programs were noted
as potential candidates for regulation of gene expression of NAP1L1 and can be seen in
Figure 1. The colors of the boxes were used to group TFBSs that were similar between
two or more of the bioinformatics programs: red – Alibaba2 and Match, blue – Alibaba2
and PROMO, green – Match and PROMO, and yellow – all three programs. The
transcription start site is indicated by a bent arrow with the label “+1”. Locations of
deletion constructs are denoted by black corners labeled with the nucleotide at the 5’ end
of each deletion construct.
30 Figure 1. Bioinformatics results of transcription factor binding sites within the promoter region of NAP1L1. Boxes
surround consensus sequences of transcription factor binding sites (TFBSs) and the transcription factor identity
labeled above or below the box. Red boxes indicate TFBSs common between the results of Alibaba2 and Match. Blue
boxes indicate TFBSs common between the results of Alibaba2 and PROMO. Green boxes indicate TFBSs common
between the results of Match and PROMO. Yellow-orange boxes indicate TFBSs common between the results of all
31 three bioinformatics programs. The transcription start site is indicated by an arrow and “+1”. Black corners with
numbers indicate the positions where the promoter region was cut to make deletion constructs.
3.2 Cloning the NAP1L1 promoter region into pGL3 Basic.
A decision was made to clone a larger region of the promoter region into pGL3
Basic as the originally cloned fragment only contained -563 bp of the sequence upstream
of the transcription start site. This new fragment includes upstream sequence up to -1273
bp 5’ of the transcription start site. An expected fragment size of 1945 bp from the initial
touchdown PCR reaction was confirmed by gel electrophoresis, which showed a bright
band just below the 2000 bp marker band. Other bands were visible on the gel but were
smaller and much more faint. After this band was excised, purified, ligated into pGC
Blue, and transformed, six of the resulting colonies were grown overnight in liquid LB
supplement with kanamycin and purified by boiling lysis. These boil preps were digested
with EcoRI to determine if the promoter region had been successfully ligated into pGC
Blue and would result in band sizes of 2233 bp and 1963 bp. A second digest with SpeI
would determine if the promoter region had been inserted in the “forward” (2505 bp and
1691 bp) or “reverse” (3874 bp and 322 bp). The “forward” direction refers to the
promoter region pointing towards the XhoI restriction site in pGC Blue. All six colonies
contained the promoter region with two in the reverse direction and four in the forward
direction. One of the samples containing the promoter in the reverse direction was then
cut with HindIII and XhoI in order to directionally clone the promoter region into pGL3
Basic to drive the expression of luciferase. After pGC Blue with the NAP1L1 promoter
region and pGL3 Basic were cut with HindIII and XhoI, the fragments were excised from
a gel, purified, and ligated together. Of the resulting colonies, six were grown overnight
32 in liquid LB supplemented with ampicillin and purified by boiling lysis. These boil preps
were digested with EcoRI to determine if the promoter region had been successfully
ligated into pGL3 Basic and would result in band sizes of 4879 bp and 1963 bp. A second
digest with NotI determined if the promoter region was inserted in the direction pointing
towards the luciferase gene (6637 bp and 205 bp) or away from the luciferase gene. Only
one of the EcoRI digests resulted in two visible bands near the 5000 bp and 2000 bp
marker bands and the NotI digest appeared to not cut at all and may not have worked.
The remaining bacterial culture containing the plasmid the resulted in the correct bands
for the EcoRI digest was used to streak a bacterial plate from which three colonies were
then grown overnight in liquid LB containing ampicillin. These cultures were then
purified using Thermo Scientific’s GeneJET Plasmid Miniprep Kit and digested with
EcoRI (4879 bp and 1963 bp), SacI (4784 bp, 2005 bp and 53 bp), and SpeI (6520 bp and
322 bp) to confirm that pGL3 Basic contained the promoter region in the correct
orientation. All three plasmids contained the correct band sizes for all three digests. The
bands in the EcoRI digests were just below the 5000 bp and just below the 2000 bp
marker bands. The bands in the SacI digests were a little bit below the 5000 bp marker
band and just above the 2000 bp marker band. The bands in the SpeI digests were
between the 6000 bp and 8000 bpmarker bands (closer to the 6000 bp marker band) and
between the 250 bp and 500 bp marker bands (closer to the 250 bp marker band). The
final recombined plasmid still contained part of the first intron that was amplified in the
original PCR reaction and needed to be removed before transfection.
33 3.3 Removing the intron from pGL3 Basic containing the NAP1L1 promoter region.
In order to remove the intron from the promoter region, the recombined vector
containing the intron was digested with SpeI. Two existing restriction sites were already
in the recombined vector, one within the small section of pGC Blue that came over when
the promoter was removed from pGC Blue and cloned into pGL3 Basic and one at the
very end of the first exon ~5 bp before the beginning of the first intron. The SpeI
restriction site in pGC Blue was conveniently on the 3’ end of the promoter fragment
removed from pGC Blue and so the pGL3 Basic vector containing the NAP1L1 promoter
region was simply cut with SpeI and ligated onto itself to remove the intron fragment.
After the ligation was transformed, 6 of the resulting colonies were set to grow overnight
in liquid LB containing ampicillin and the plasmids were purified by boiling lysis. The
purified plasmids were digested with KpnI and XbaI to confirm the removal of the intron
fragment. Expected band sizes from the KpnI digest were 4772 bp and 1748 bp and from
the XbaI digest were 4169 bp and 2351 bp. Two of the boil preps were cut correctly by
EcoRI and XbaI. The EcoRI digests resulted in two bands, one between the 4000 bp and
5000 bp marker bands (closer to 5000 bp band) and one centered between the 1500 bp
and 2000 bp marker bands. The XbaI digests results in two bands as well, one between
the 4000 bp and 5000 bp marker bands (closer to the 4000 bp band) and one between the
2000 bp and 2500 bp marker bands (closer to the 2500 bp band). The final, correct
plasmid, now referred to as pGL3N-1273, was then used for transfections, for the intron
(as a potential enhancer) cloning, and for the remaining promoter deletion constructs (398, -367, -335, -254, and -215).
34 3.4 Cloning the intron into the enhancer region of pGL3 Basic and pGL3N-1273.
In order to determine if there is a functional enhancer within the first intron of
NAP1L1, a region of 1913 bp was amplified by touchdown PCR. This fragment, which
contained almost the entire first exon in addition to a region of the first exon, appeared
just below the 2000 bp marker band as expected. One other faint band was visible. The
fragment was excised, purified, ligated into pGC Blue, and transformed. Twelve of the
resulting colonies were set up in liquid LB supplemented with kanamycin overnight and
the plasmids were purified through boiling lysis. These purified plasmids were digested
with EcoRI to confirm that the intron region had been cloned into pGC Blue. The EcoRI
digests were expected to result in three bands at 2233 bp, 1045 bp, and 886 bp. Of the 12
colonies, 11 had plasmids that resulted in the correct band sizes. To determine the
orientation of these plasmids, 6 were chosen and digested with KpnI. The KpnI digests
were expected to result in two bands at 3396 bp and 768 bp if in the “reverse” direction
and two bands at 2915 bp and 1249 bp if in the “forward” direction. Of these, 5 were in
the reverse direction with a band between the 3000 bp and 3500 bp marker bands (closer
to 3500 bp) and a band at the 750 bp marker band. The other plasmid contained the intron
in the forward direction with one band just beneath the 3000 bp marker band and one
band midway between the 1000 bp and 1500 bp marker band. One of the plasmids
containing the intron in the reverse direction was then digested with SpeI to remove the
intron for cloning into pGL3 Basic and pGL3N-1273 while at the same time remove the
majority of the 5’-UTR that was amplified in the original PCR. Both pGL3 Basic and
pGL3N-1273 were digested with SalI, which cuts 3’ of the luciferase coding region. All
three linearized fragments were blunted and the two vectors were treated with CIP to
35 remove the phosphates. All three were run on a gel, excised, purified, ligated, and
transformed. Ten colonies from the plate of pGL3 Basic with the intron and 6 colonies
from the plate of pGL3N-1273 with the intron were grown overnight in liquid LB
containing ampicillin and the plasmids were purified by boiling lysis. The purified
plasmids were digested with EcoRI to determine both if the intron had been successfully
inserted into the vectors and in which direction the intron had been inserted.
3.5 Transient transfections of NAP1L1 promoter deletion constructs revealed
regions between -442 bp and -297 bp that may contain a positive regulatory element
and -297 bp and -183 bp that may contain a negative regulatory element.
In order to determine the locations of regulatory elements within the isolated
promoter region of NAP1L1, initial deletions of the promoter region in pGL3 Basic were
transfected into human embryonic kidney (HEK293T) cells alongside the full promoter
region. In order to test these promoter constructs for their ability to drive the expression
of luciferase, the constructs were co-transfected with the normalization vector pRLSV40. After normalization of firefly luciferase expression from the pGL3 constructs to
the Renilla luciferase expression from pRL-SV40, a fold value was calculated based on
the minimum expression of luciferase by the negative control, pGL3 Basic. The pGL3
Basic vector (Promega) contains no promoter region and so resulted in the least amount
of luciferase expression. One positive control was used as well, pGL3 Control, which
contains the SV40 promoter and enhancer (blue box in Figure 2) (Promega). The controls
pGL3 Basic and pGL3 Control are the first two vectors from the top in Figure 2. The
luciferase coding region is denoted by a yellow box labeled “luc+”. All NAP1L1
36 promoter constructs contain a 5’–UTR (purple bar) that extends to +387 relative to the
transcription start site upstream of luc+, a transcription start site (bent arrow), and an
upstream promoter region (black bar) labeled with the distance from the transcription
start site. Luciferase activity resulting from the construct containing the NAP1L1
promoter region (pGL3N-1273), spanning from -1273 bp to +387 bp relative to the
transcription start site (+1), showed high luciferase activity similar to that of the pGL3
Control vector (Figure 2). Little differences in luciferase activity were seen between
pGL3N-1273, pGL3N-563, and pGL3N-442 (Figure 2). However, a drop in luciferase
activity was seen between the pGL3N-442 and pGL3N-297 constructs (Figure 2).
Although less apparent in the figure, there is a small increase in luciferase activity
between the pGL3N-297 and pGL3N-183 constructs. The somewhat larger increase in
luciferase activity between pGL3N-183 and pGL3N-56 was inconsistent between
transfections and so produced large error bars. Although the error bars in this initial
experiment are large, there was a consistent pattern between transfections that resulted in
a drop in luciferase activity between pGL3N-442 and pGL3N-297 and an increase in
37 luciferase activity between pGL3N-297 and pGL3N-183, and so was a basis for
designing smaller deletions within these two regions.
Figure 2. Luciferase activity resulting from promoter deletion constructs. Fold values were calculated by
normalizing to the expression results of pGL3 Basic, the luciferase reporter vector containing no promoter.
The positive control vector pGL3 Control contains an SV40 promoter and enhancer, show an increase in
luciferase activity relative to pGL3 Basic. The largest isolated NAP1L1 promoter region, pGL3N-1273,
shows a high amount of luciferase activity similar to the pGL3 Control vector. The first notable change in
luciferase activity occurs between the pGL3N-442 and pGL3N-297 vectors, where a large decrease in
luciferase activity is seen in the pGL3N-297 construct. There also appears to be an increase in luciferase
activity between the pGL3N-183 and pGL3N-56 constructs although the error bars are large. n=3, Mean ±
S.D.
38 3.6 Preliminary transient transfections results from the smaller NAP1L1 promoter
deletions between -442 bp and -297 bp as well as between -297 bp and -183 bp
relative to the TSS.
Smaller deletions within the two regions between -442 bp and -183 bp were made
to narrow down the locations of potential regulatory elements. The constructs pGL3N442, pGL3N-398, pGL3N-367, pGL3N-335, pGL3N-297, pGL3N-254, pGL3N-215, and
pGL3N-183 were transfected into HEK293T cells along with the controls pGL3 Basic
and pGL3 Control and the full promoter construct pGL3N-1273. Preliminary transfection
results are shown in Figure 3 below. The first three constructs are the control vectors as
previously described above. The following promoter constructs are also pictured as
previously described above. Given this preliminary data, there appears to be a decline in
luciferase activity between the pGL3N-367 and pGL3N-335 constructs and two increases
in luciferase activity between constructs pGL3N-297 and pGL3N-215 (Figure 3). The
first difference is seen between pGL3N-297 and pGL3N-254 and the second between
pGL3N-254 and pGL3N-215 (Figure 3).
39 Figure 3. Preliminary results from transient transfections of smaller deletions within the region -442 bp and -183 bp
relative to the transcription start site. The controls are the top three constructs in the graph. The first is pGL3 Basic,
which contains no promoter. The third is pGL3 Control, which contains the SV40 promoter and enhancer (blue box). In
the construct below, the 5’ upstream sequence of the promoter region is denoted by the black bar and number of the
most 5’ nucleotide of the promoter region. The transcription start site is denoted by the bent arrow and the 5’-UTR is
denoted by the purple bar that ends at +387 bp in all constructs. The yellow box indicates the coding region for the
firefly luciferase gene in the pGL3 constructs. This preliminary data shows a decrease in luciferase activity between 367 bp and -335 bp relative to the transcription start site and an increase in luciferase activity between -297 bp and 254 bp relative to the transcription start site. There may be a second increase in luciferase activity between the
pGL3N-254 and pGL3N-215 constructs as well. n=2, Mean ± S.D.
40 3.7 Transient transfections of the NAP1L1 promoter and intron regions.
In order to determine whether or not an enhancer lies within the first intron of
NAP1L1, as was seen in mice (Wu et al., 2008), a section of the first intron spanning
from +382 bp to +1915 bp was cloned into the region 3’ of the luciferase coding
sequence in pGL3 Basic and pGL3N-1273. Controls pGL3 Basic and pGL3 Control were
used in this set of transfections along with a recombined pGL3 Basic (no promoter)
containing the intron in the forward and the reverse directions. The vectors pGL3N-1273,
pGL3N-1273 + intron(F), and pGL3N-1273 + intron(R) were transfected to determine if
the intron contained a functional enhancer. The images in Figure 4 are as previously
described, except the included intron region is denoted as a green box with a green arrow
indicating its orientation and the putative c-Myc binding site is denoted as a brownish
oval. Luciferase activity of the pGL3 Basic constructs (no promoter) with the intron in
both directions or without the intron were similar as expected (Figure 4). These initial
results show a large increase in luciferase activity resulting from the pGL3N-1273
construct containing the intron in the forward orientation compared to the promoter
construct without the intron, pGL3N-1273 (Figure 4). There seemed to be no difference
in luciferase activity resulting from the construct containing the intron in the reverse
41 direction compared to the construct containing only the full promoter region, pGL3N1273 (Figure 4).
Figure 4. Initial transfections of NAP1L1 intron constructs suggest that a function enhancer lies within the first
intron of NAP1L1. Control constructs are depicted as previously described. Intron contructs contain the intron region
(green box) and an arrow (green) representing the orientation of the intron within the construct. The putative c-Myc
binding site is illustrated as a brownish oval within the intron. Results show a large increase in luciferase activity
between the NAP1L1 promoter construct and the NAP1L1 promoter construct containing the intron region in the
forward direction. The NAP1L1 promoter construct containing the intron in the reverse direction did not seem to result
in a difference in luciferase activity compared to the NAP1L1 promoter construct without an intron. n=3, Mean ± S.D.
3.8 Transient transfections of NAP1L1 promoter and intron regions with the
mutated c-Myc consensus sequence.
To determine if the c-Myc consensus sequence within the first intron of NAP1L1
is involved in the expression of NAP1L1, the consensus sequence was mutated in the
promoter construct pGL3N-1273 containing the intron in the forward direction from
“CACGTG” to “TTTGTG”. This mutated construct, depicted in Figure 5 as previously
42 described except for a red “X” over the putative c-Myc binding site, was transfected
alongside the initial set of intron constructs (see 3.7). The results show that mutating the
c-Myc consensus sequence within the intron decreased the amount of luciferase activity
to a fold value not significantly different from the NAP1L1 promoter construct without an
intron (Figure 5). There is a significant difference in luciferase expression between the
promoter construct without the intron region and the one with the intron in the forward
direction (Figure 5). There was no significant difference between the promoter construct
without the intron and the promoter construct with the intron in the reverse direction
(Figure 5). There was a significant difference in luciferase expression between the
promoter construct with the intron in the forward direction and the same construct
containing the mutation at the putative c-Myc site (Figure 5). The construct containing
the promoter of NAP1L1 and the intron in the reverse direction showed a significant
difference in luciferase expression from the NAP1L1 promoter construct containing the
mutated intron in the forward direction (Figure 5). An unpaired student’s t-test was used
to determine the significance between the results of two constructs at a time.
43 Figure 5. Transient transfections of the NAP1L1 intron constructs including the c-Myc binding site mutation. The
controls are the same as those previously described. showed increases in luciferase activity relative to the pGL3 Basic
vector. The controls pGL3 Basic containing the intron in the forward direction and pGL3 Basic containing the intron
in the reverse direction resulted in low levels of luciferase activity similar to the empty pGL3 Basic vector. The
construct pGL3N-1273 containing the intron in the forward direction resulted a large increase in luciferase activity,
approximately twice that of the construct containing only the NAP1L1 promoter region. The construct containing the
NAP1L1 promoter region and the intron in the reverse direction, showed similar luciferase activity to that of the
pGL3N-1273 construct. The promoter construct containing the NAP1L1 promoter region and the intron in the forward
orientation with the mutated sequence “TTTGTG” resulted in a decrease in luciferase activity relative to the nonmutated construct. This mutated construct resulted in similar levels of luciferase activity to the pGL3N-1273 construct
that did not contain an intron. n=3, Mean ± S.D., *p<0.05.
44 CHAPTER 4: DISCUSSION
Gene expression is modulated by specific transcription factors bound to
sequences, such as proximal promoter elements, enhancers, and silencers, that interact
with basal transcription factors at the core promoter to increase or decrease transcription
(Maston et al., 2006). Much of the differential regulation between cell types comes from
the binding of specific transcription factors to proximal promoter elements, enhancers,
and silencers. In order to better understand how a particular gene is regulated in a normal
cell, these differential elements within the promoters and distal elements must be studied.
4.1 Transient transfections of initial deletion constructs revealed a regulatory
element located within the region between -442 bp and -297 bp relative to the
transcription start site (+1) and smaller deletions of this region suggest that the
element lies between -367 bp and -335 bp relative to the TSS.
Transient transfections of larger deletion constructs resulted in a decrease in
luciferase activity between the pGL3N-442 and pGL3N-297 constructs, suggesting that a
positive regulatory element lies between -442 bp and -297 bp relative to the transcription
start site (Figure 2). The preliminary results from the transfections with the smaller
deletions within this region suggests that the element lies between -367 bp and -335 bp
relative to the transcription start site (Figure 3). Bioinformatics analyses did not suggest a
particular transcription factor binding site within this region (Figure 1). Results from
individual bioinformatics programs (Alibaba2, Match, and PROMO) suggest the
sequence contains bindings sites for transcription factors such as C/EBPb, XBP-1,
45 RREB-1, HNF-4, Sp1, and others (data not shown). These five transcription factors are
capable of functioning as activators. XBP-1 has been shown to bind the promoter of
GPR43 and upregulate its expression in human monocytes (Ang et al., 2015). RREB-1
has both activating and suppressing capabilities (Jiang et al., 2010; Milon et al., 2010).
When the binding site for RREB-1 is deleted in the promoter of TCblR in a luciferase
reporter transfected into HEK293T cells, the luciferase activity decreases significantly
(Jiang et al., 2010). HNF-4a increases the expression of CLDN7 in Caco-2 cells (Farkas
et al., 2015) and increases the expression of EPXH1 with other factors in HepG2 cells
(Peng et al., 2015). Sp1 is known to have roles in activating or suppressing transcription
(Li et al., 2015; Zaid et al., 2001). For example, as an activator Sp1 was shown to bind
and upregulate MALAT1 in human cells (Li et al., 2015). In order to determine which
transcription factor is binding this region, more information will need to be obtained
through luciferase assays of sequence mutations.
4.2 Transient transfections of deletion constructs revealed a regulatory element
located within the region between -297 bp and -183 bp relative to the transcription
start site (+1) and smaller deletions of this region suggest that there is an element
that lies between -297 bp and -254 bp and a second element that lies between -254
bp and -215 bp relative to the TSS.
Results from preliminary luciferase assays revealed an increase in luciferase
expression between the constructs pGL3N-297 and pGL3N-254, suggesting that a
negative regulatory element lies between -297 bp and -254 bp. The results from the
bioinformatics analyses (Figure 3) do not show a potential TFBS in this region common
46 between the bioinformatics programs. The individual programs (Alibaba2, Match, and
PROMO) suggest transcription factor binding sites such as GRa, C/EBPb, TGIF, Sp1,
Elf-1, and others (data not shown). GR, in mice and humans, may function as a
transrepressor by interacting with other factors to inhibit the expression of a gene (e.g.
AP-1) instead of binding the DNA sequence directly (De Bosscher et al., 2001; Newton,
2014). Sp1 acts as a repressor of ANT2 in Hela and NIH3T3 cells (Zaid et al., 2001). Elf1 may function to upregulate or downregulate gene expression (Xiang et al., 2010; Honda
et al., 2003). In rats, the bidirectional promoters for Nth1 and Tsc2 are suppressed by Elf1 binding to two sequences in between these promoters that are conserved in humans
(Honda et al., 2003). Luciferase assays of sequence mutations within this small region of
the NAP1L1 promoter will help identify which specific sequences are involved in gene
regulation and then sequence binding by a specific transcription factor can then be tested
via a ChIP analysis.
There also seems to be an increase in luciferase expression between the constructs
pGL3N-254 and pGL3N-215, also suggesting the location of a negative regulatory
element within the region -254 bp to -215 bp relative to the TSS. The bioinformatics data
in Figure 1 shows a putative transcription factor binding site for the progesterone receptor
(PR) in this region. The two isoforms of PR, PR-A and PR-B, have activating
capabilities, but it seems that PR-A may have suppressive capabilities as well (Patel et
al., 2015). This is a tentative result that will need support from experimental data by
luciferase assays of smaller deletions or sequence mutations within this region.
47 4.3 Transient transfections of initial deletion constructs revealed a potential element
between -183 bp and -56 bp relative to the TSS.
The rather large increase in luciferase expression between constructs pGL3N-183
and pGL3N-56 was variable between transfections, but may need to be looked into
further for potential regulatory elements. As this construct encompasses the core
promoter and a little upstream sequence, there may be downstream regulatory elements
that are involved in this high expression level. Based on the bioinformatics data in Figure
1, this region contains two putative Sp1 binding sites and two AP-2a binding sites, one of
which overlaps the junction at -56 bp. Ap-2a has activating capabilities as well as
silencing capabilities, as it is known to act as a repressor in mice (Jiang et al., 1998,
Berlato et al., 2011). These speculations based on the bioinformatics data will need to be
tested experimentally through luciferase assays of smaller deletion constructs or sequence
mutations within this region.
4.4 Transfections of intron constructs revealed a functional c-Myc binding site in the
first intron of NAP1L1.
Wu and colleagues (2008) identified c-Myc binding sites within the first intron of
murine NAP1L1 after a ChIP-seq of c-Myc that brought down the promoter region
containing part of the first intron with two c-Myc consensus sequences. In a follow-up
experiment, a strong correlation was made between high levels of c-Myc and NAP1L1 in
human B cell lymphomas (Wu et al., 2008). This led to the idea that there is a conserved
c-Myc binding site within the first intron of the human NAP1L1. In order to determine if
48 there is a functional enhancer within the first intron of NAP1L1, a region of the intron
was cloned into pGL3N-1273 with the expectation of seeing an increase in gene
expression. An increase in luciferase activity seen in the construct containing the intron in
the forward direction supports the hypothesis that there is an enhancer within the first
intron of NAP1L1. Enhancers are generally known to function independent of their
location relative to the promoter region (Maston et al., 2006). They are bound by
activating transcription factors and form an enhanceosome complex at the promoter of a
gene by looping around to interact with the transcription factors bound to the promoter
(Maston et al., 2006; Engel et al., 2016). As enhancers were previously characterized as
functioning in both a location- and orientation-independent manner (Maston et al., 2006),
the intron was cloned into a region further away from the NAP1L1 promoter (on the other
side of the luciferase coding region) and a second construct with the intron in the reverse
direction was made. Interestingly, and unexpectedly, an increase was not seen in the
construct containing the intron in the reverse direction, suggesting the enhancer may be
working in an orientation-dependent manner. The oncogene MYC produces a
transcription factor c-Myc that appears to be a regulator of NAP1L1 in mice based on the
results of a c-Myc ChIP-seq (Wu et al., 2008). The c-Myc consensus sequence in the first
intron of human NAP1L1 may be functionally conserved as there appears to be an
increase in luciferase expression with the construct containing the intron region in the
forward direction. The displacement of this binding site is minimal between the forward
and reverse orientations, shifting the location of the consensus sequence by
approximately 300 bp. If found to be a regulator of NAP1L1 expression in humans, cMyc would seem to function to increase gene expression in a location-independent but
49 orientation-dependent manner. There is some evidence of orientation-dependent enhancer
elements. One example, in mice, shows that an enhancer in between two divergent
promoters acts in an orientation-dependent manner (Swamynathan and Piatigorsky,
2002). When this enhancer was inverted in the luciferase construct, the promoter of one
of the genes resulted in a decrease of luciferase expression to ~6% that of the wild-type
promoter. Another example, also previously mentioned, is the enhancer that regulates the
yellow gene in Drosophila melanogaster but not in a closely related species that contains
an inverted consensus sequence (Jeong et al., 2006). Although the intron region of
NAP1L1 does not appear to function in an orientation-dependent manner, as was
previously thought to be characteristic of enhancers (Engel et al., 2016), the pGL3N1273 construct containing the intron in the forward direction did result in increased
luciferase activity relative to the pGL3N-1273 construct without the intron region. There
should also be some caution in the interpretation of these results, as the location and
orientation of the enhancer within these constructs does not reflect that which is in the
living cell and so some results, such as the decreased luciferase activity from the
construct containing the inverted intron, may be a consequence of in vitro experiments.
After the intron was shown to increase gene expression within the plasmid
containing the full, isolated promoter region of NAP1L1 and the intron in the forward
direction, the putative c-Myc binding site within the intron was tested for its potential
involvement as an enhancer. The consensus sequence for c-Myc was mutated from
“CACGTG” to “TTTGTG” in the full pGL3N-1273 construct containing the intron in the
forward direction. The resulting luciferase activity was comparable to that of the full
promoter construct without the intron (pGL3N-1273) (Figure 5). This confirmed the
50 hypothesis that the sequence “CACGTG” is important in the expression of NAP1L1. It is
known that c-Myc and Max form a heterodimer to bind the sequence, called an E-box
(Walhout et al., 1997).
Regulatory factors in addition to c-Myc such as the upstream stimulating factor
(USF) have been shown to bind the E-box sequence (Walhout et al., 1997). Although
there are other factors that bind the same sequence and regulate other genes containing
that sequence, c-Myc seems to be the most likely transcription factor to regulate NAP1L1
based on the previous research by Wu and colleagues (2008), indicating a correlation
between high levels of NAP1L1 expression and c-Myc in human lymphomas. c-Myc has
been found to regulate other genes via an intronic enhancer including the rat ornithine
decarboxylase (ODC) (Walhout et al., 1997) and rat prothymosin a (Gaubatz et al.,
1994). In fact, intronic enhancers are not uncommon. There are examples of functional
enhancers within introns of human genes such as FDXR and GCH, and murine genes
such as GnRH and NAP1L1 (Imamichi et al., 2014; Liang et al., 2013; Kim et al., 2011;
Wu et al., 2008).
If c-Myc is found to be a regulator of NAP1L1 expression, this could reveal a
method by which NAP1L1 is overexpressed in certain cancers such as pancreatic
neuroendocrine neoplasms and small intestinal carcinoids (Kidd et al., 2006; Schimmack
et al., 2014). As c-Myc is a known oncogene and NAP1L1 overexpression has been
correlated with cancer, the regulation of NAP1L1 by c-Myc may suggest a method by
which c-Myc overexpression results in tumor growth. The overexpression of NAP1L1 has
been correlated with the ability of induced pluripotent stem cells to maintain a
proliferative and a stem-like state instead of differentiating (Gong et al., 2014). A recent
51 study showed that when NAP1L1 expression is knocked down in murine iPSCs, there was
an increase in differentiation into cardiomyocytes (Gong et al., 2014). This same study
showed that overexpression of NAP1L1 decreased iPSC differentiation into
cardiomyocytes. A more recent study by the same lab confirmed NAP1L1’s role in the
proliferative ability of murine iPSCs through knockdown and overexpression
experiments (Yan et al., 2016). It is noteworthy that c-Myc is one of the four Yamanaka
factors involved in inducing pluripotency (Yan et al., 2016), which may, in part, result
from direct upregulation of NAP1L1 through its intronic enhancer as is seen in mice (Wu
et al., 2008) and humans (this study). NAP1L1 overexpression has also been correlated
with multiple cancers such as small intestinal carcinoids (SICs), hepatoblastomas, and
pancreatic neuroendocrine neoplasms (Kidd et al., 2006; Nagata et al., 2003; Schimmack
et al., 2014). This increased expression may be a result of upregulation by c-Myc, which
is known to be expressed at higher levels in many cancers (Stine et al., 2015; Udager et
al., 2016). This may suggest a potential method of downregulating NAP1L1 as a cancer
treatment or in order to differentiate stem cells into specific tissues for grafting. As
methods such as CRISPR-Cas9 are advancing and approaching clinical use, there may
arise a method to downregulate NAP1L1 by mutating the c-Myc binding site within the
first intron of the human NAP1L1 in order to decrease proliferation in cancer or increase
differentiation in iPSCs for treatment (Sánchez-Rivera and Jacks, 2015; Sternberg and
Doudna, 2015).
52 4.5 Future directions.
After confirming that the sequence “CACGTG” within the first intron is involved
in the expression of NAP1L1, the next step would be a ChIP assay to confirm whether or
not it is the transcription factor c-Myc that is binding this sequence.
Given the preliminary data from the small promoter deletions between -442 bp
and -183 bp relative to the TSS, a potential transcription factor binding site within the
regions -367 bp and -335 bp relative to the TSS and -297 bp and -215 bp relative to the
TSS will be mutated to determine if there is a similar change in luciferase activity. As the
bioinformatics data did not collectively suggest a specific transcription factor binding site
within this region, sequence mutations within each of these regions can be made in order
to determine specific sequences that are regulating gene expression. A ChIP assay will
then confirm whether or not the putative transcription factor binds that sequence.
Transient transfections of smaller deletions within the region -183 bp to -56 bp relative to
the TSS will also be necessary in order to reveal potential elements within this region.
Identifying the mechanisms of gene regulation in normal, healthy cells can help
reveal the methods in which dysregulation of certain genes occurs in diseases such as
cancer. For example, tumors can form from uncontrollably dividing cells whose cell
cycle genes and apoptotic genes are improperly regulated. Developmental disorders can
result from improper gene regulation as well and the effects will depend on what genes
and at which stage the dysregulation occurs.
The nucleosome assembly protein 1-like 1 (NAP1L1) has been associated with
multiple cancers as well as the stem-like nature of undifferentiated cells. When the
53 expression of this gene was knocked down in murine pancreatic neuroendocrine
neoplasms, the proliferation of these cells decreased (Schimmack et al., 2014). When this
gene’s expression was knocked down in murine iPSCs, the cells proceeded to
differentiate into cardiomyocytes (Gong et al., 2014). Studying the factors involved in the
expression of this gene is important for understanding why this gene is expressed
differently between the two B-cell lymphomas, mantle cell lymphoma and small
lymphocytic lymphoma (Henson et al., 2011).
Studying how genes are expressed allows for a better understanding of diseases
caused by the dysregulation of genes as well as a better understanding of gene expression
in general.
54 REFERENCES
Ang Z, Er JZ, Ding JL. The short-chain fatty acid receptor GPR43 is transcriptionally
regulated by XBP1 in human monocytes. Nature Scientific Reports 2015; doi:
10.1038/srep08134
Attia M, Förster A, Rachez C, Freemont P, Avner P, Rogner UC. Interaction between
Nucleosome Assembly Protein 1-like Family Members. Journal of Molecular Biology
2011;407:647-660.
Berlato C, Chan KV, Price AM, Canosa M, Scibetta AG, Hurst HC. Alternative TFAP2A
isoforms have distinct activities in breast cancer. Breast Cancer Research 2011;13:R23.
Danino YM, Even D, Ideses D, Juven-Gershon T. The core promoter: At the heart of
gene expression. Biochimica et Biophysica Acta 2015;1849:1116-1131.
De Bosscher K, Berghe WV, Haegeman G. Glucocorticoid Repression of AP-1 Is Not
Mediated by Competition for Nuclear Coactivators. Molecular Endocrinology
2001;15:219–227.
Dikstein R. The unexpected traits associated with core promoter elements. Transcription
2011;2:201-206.
Engel KL, Mackiewicz M, Hardigan AA, Myers RM, Savic D. Decoding transcriptional
enhancers: Evolving from annotation to functional interpretation. Seminars in Cell &
Developmental Biology 2016;57:40-50.
Farkas AE, Hilgarth RS, Capaldo CT, Gerner-Smidt C, Powell DR, Vertino PM, Koval
M, Parkos CA, Nusrat A. HNF4a Regulates Claudin-7 Protein Expression during
Intestinal Epithelial Differentiation. The American Journal of Pathology 2015;185:2206–
2218.
Gaubatz S, Meichle A, Eilers M. An E-Box Element Localized in the First Intron
Mediates Regulation of the Prothymosin a Gene by c-myc. Molecular and Cellular
Biology 1994;14:3853–3862.
Gehrke AR, Shubin NH. Cis-regulatory programs in the development and evolution of
vertebrate paired appendages. Seminars in Cell & Developmental Biology 2016;57:31–
39.
Gong H, Yan Y, Fang B, Xue Y, Yin P, Li L, Zhang G, Sun X, Chen Z, Ma H, Yang C,
Ding Y, Yong Y, Zhu Y, Yang H, Komuro I, Ge J, Zou Y. Knockdown of Nucleosome
Assembly Protein 1-Like 1 Induces Mesoderm Formation and Cardiomyogenesis Via
Notch Signaling in Murine-Induced Pluripotent Stem Cells. Stem Cells 2014;32:1759–
1773.
55 Goodrich JA, Tjian R. Unexpected Roles for Core Promoter Recognition Factors in Celltype Specific Transcription and Gene Regulation. Nat Rev Genet. 2010;11:549-558.
Haberle V, Lenhard B. Promoter architectures and developmental gene regulation.
Seminars in Cell & Developmental Biology 2016;57:11-23.
Henson SE, Morford T, Stein MP, Wall R, Malone CS. Candidate genes
contributing to the aggressive phenotype of mantle cell lymphoma. Acta
Histochemica 2011;113:729–742.
Honda S, Kobayashi T, Kajino K, Urakami S, Igawa M, Hino O. Ets Protein Elf-1
Bidirectionally Suppresses Transcriptional Activities of the Tumor Suppressor
Tsc2 Gene and the Repair-Related Nth1 Gene. Molecular Carcinogenesis
2003;37:122–129.
Imamichi Y, Mizutani T, Ju Y, Matsumura T, Kawabe S, Kanno M Yazawa T,
Miyamoto K. Transcriptional regulation of human ferredoxin reductase through
an intronic enhancer in steroidogenic cells. Biochimica et Biophysica Acta
2014;1839:33–42.
Jeong S, Rokas A, Carroll SB. Regulation of Body Pigmentation by the
Abdominal-B Hox Protein and Its Gain and Loss in Drosophila Evolution. Cell
2006;125:1387–1399.
Jiang MS, Tang QQ, McLenithan J, Geiman D, Shillinglaw W, Henzel WJ, Lane
MD. Derepression of the C/EBPa gene during adipogenesis: Identification of AP2a as a repressor. Proc. Natl. Acad. Sci. 1998;95:3467–3471.
Jiang W, Sequeira JM, Nakayama Y, Quadros EV. Characterization of the
promoter region of TCblR/CD320 gene, the receptor for cellular uptake of
transcobalamin-bound cobalamin. Gene 2010;466:49–55.
Kidd M, Modlin IM, Mane SM, Camp RL, Eick G, Latich I. The role of genetic
markers — NAP1L1, MAGE-D2, and MTA1 — in defining small-intestinal
carcinoid neoplasia. Annals of Surgical Oncology 2006;13:253–262.
Kim, HD, Choe HK, Chung S, Kim M, Seong JY, Son GH, Kim K. Class-C SOX
Transcription Factors Control GnRH Gene Expression via the Intronic
Transcriptional Enhancer. Mol Endocrinol 2011;25:1184–1196.
Kim MH, Field J. Translationally Regulated C/EBPb Isoform Expression
Upregulates Metastatic Genes in Hormone-Independent Prostate Cancer Cells.
The Prostate 2008;68:1362–1371.
56 Li S, Wang Q, Qiang Q, Shan H, Shi M, Chen B, Zhao S, Yuan L. Sp1-mediated
transcriptional regulation of MALAT1 plays a critical role in tumor. J Cancer Res
Clin Oncol 2015;141:1909–1920.
Liang Y, Inagaki H, Hao Q, Sakamoto M, Ohye T, Suzuki T, Ichinose H.
Identification of an enhancer region for immune activation in the human GTP
cyclohydrolase I gene. Biochemical and Biophysical Research Communications
2013;442:72–78.
Maston GA, Evans SK, Green MR. Transcriptional Regulatory Elements in the
Human Genome. Annual Review of Genomics and Human Genetics 2006;7:2959.
Milon BC, Agyapong A, Bautista R, Costello LC, Franklin RB. Ras Responsive
Element Binding Protein-1 (RREB-1) Down-Regulates hZIP1 Expression in
Prostate Cancer Cells. Prostate 2010;70:288–296.
Mishra S, Lin CL, Huang TH, Bouamar H, Sun LZ. MicroRNA-21 inhibits
p57Kip2 expression in prostate cancer. Mol Cancer 2014;13:212.
Nagata T, Takahashi Y, Ishii Y, Asai S, Nishida Y, Murata A, Koshinaga T,
Fukuzawa M, Hamazaki M, Asami K, Ito E, Ikeda H, Takamatsu H, Koike K,
Kikuta A, Kuroiwa M, Watanabe A, Kosaka Y, Fujita H, Miyake M, Mugishima
H. Transcriptional profiling in hepatoblastomas using high-density
oligonucleotide DNA array. Cancer Genetics and Cytogenetics 2003;145:152–
160.
Newton R. Anti-inflammatory glucocorticoids: Changing concepts. European
Journal of Pharmacology 2014;724:231–236.
Okuwaki M, Kato K, Nagata K. Functional characterization of human
nucleosome assembly protein 1-like proteins as histone chaperones. Genes to
Cells 2010;15:13-27.
Park YJ, Luger K. Structure and function of nucleosome assembly proteins.
Biochem. Cell Biol. 2006;84:549–558.
Patel B, Elguero S, Thakore S, Dahoud W, Bedaiwy M, Mesiano Sam. Role of
nuclear progesterone receptor isoforms in uterine pathophysiology. Human
Reproduction Update 2015;21:155–173.
Peng H, Zhu QS, Zhong S, Levy D. Transcription of the human microsomal
epoxide hydrolase gene (EPHX1) is regulated by an HNF-4a/CAR/RXR/PSF
complex. Biochimica et Biophysica Acta 2013;1829:1000–1009.
57 Sánchez-Rivera FJ, Jacks T. Applications of the CRISPR-Cas9 system in cancer
biology. Nat Rev Cancer. 2015;15:387–395.
Schimmack S, Taylor A, Lawrence B, Alaimo D, Schmitz-Winnenthal H, Büchler
MW, Modlin IM, Kidd M. A mechanistic role for the chromatin modulator,
NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases.
Epigenetics & Chromatin 2014;7:15.
Sternberg SH, Doudna JA. Expanding the Biologist’s Toolkit with CRISPR-Cas9.
Mol Cell. 2015;58:568–574.
Stine ZE, Walton ZE, Altman BJ, Hsieh AL, Dang CV. MYC, Metabolism, and
Cancer. Cancer Discov. 2015;5:1024–1039.
Swamynathan SK, Piatigorsky J. Orientation-dependent Influence of an Intergenic
Enhancer on the Promoter Activity of the Divergently Transcribed Mouse
Shsp/aB-crystallin and Mkbp/HspB2 Genes. The Journal of Biological Chemistry
2002;277:49700–49706.
Tu WB, Helander S, Pilstål R, Hickman KA, Lourenco C, Jurisica I, Raught B,
Wallner B, Sunnerhagen M, Penn LZ. Myc and its interactors take shape.
Biochimica et Biophysica Acta 2015;1849:469–483.
Udager AM, De Marzo AM, Shi Y, Hicks JL, Cao X, Siddiqui J, Jiang H,
Chinnaiyan AM, Mehra R. Concurrent nuclear ERG and MYC protein
overexpression defines a subset of locally advanced prostate cancer: potential
opportunities for synergistic targeted therapeutics. Prostate 2016;76:845–853.
Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: a review of
regulation, deregulation and therapeutic targets in cancer. Cell Proliferation
2003;36:131-149.
Walhout AJM, Gubbels JM, Bernards R, van der Vliet PC, Timmers HThM. cMyc/Max heterodimers bind cooperatively to the E-box sequences located in the
first intron of the rat ornithine decarboxylase (ODC) gene. Nucleic Acids
Research 1997;25:1493-1501.
Wu CH, Sahoo D, Arvanitis C, Bradon N, Dill DL, Felsher DW. Combined
Analysis of Murine and Human Microarrays and ChIP Analysis Reveals Genes
Associated with the Ability of MYC To Maintain Tumorigenesis. PLoS Genetics
2008;4:e1000090.
Xiang P, Lo C, Argiropoulos B, Lai CB, Rouhi A, Imren S, Jiang X, Mager D,
Humphries RK. Identification of E74-like factor 1 (ELF1) as a transcriptional
regulator of the Hox cofactor MEIS1. Exp Hematol. 2010;38:798–808.
58 Xu XY, Wang WQ, Zhang L, Li YM, Tang M, Jiang N, Cai SL, Wei L, Jin F,
Chen B. Clinical implications of p57 KIP2 expression in breast cancer. Asian Pac
J Cancer Prev. 2012;13:5033-5036.
Yan Y, Yin P, Gong H, Xue Y, Zhang G, Fang B, Chen Z, Li Y, Yang C, Huang
Z, Yang X, Ge J, Zou Y. Nucleosome Assembly Protein 1-Like 1 (Nap1l1)
Regulates the Proliferation of Murine Induced Pluripotent Stem Cells. Cellular
Physiology and Biochemistry 2016;38:340–350.
Zaid A, Hodny Z, Li R, Nelson BD. Sp1 acts as a repressor of the human adenine
nucleotide translocase-2 (ANT2) promoter. Eur. J. Biochem. 2001;268:5497–
5503.
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