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Developing new tools to discover and investigate histone H3 proteases

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Developing new tools to discover and investigate histone H3 proteases

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Developing New Tools to Discover and Investigate
Histone H3 Proteases

by

Shulin Liu

A Thesis Presented to the
FACULTY OF THE USC Keck School of Medicine
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
Biochemistry and Molecular Medicine

August 2020

Copyright 2020 Shulin Liu
ii

ACKNOWLEDGEMENTS
First, I want to express my thanks to my lab advisor, Dr. Judd Rice, for giving me the
chance to work in his laboratory. He provided us the most comfortable research
environment so that we could focus on our study. When I am facing difficulties, which
happened through out my project, he always discussed with me and helped me find out
the solution. As an international student, sometimes it’s hard for me to deal with
presentation or written assay alone. Dr. Rice always helped me polish the drafts and
prepare for presentations. He is also an understanding advisor that allowed us to put our
coursework first. I’m so glad to join his laboratory.
Also, I want to thank my thesis committee member, Prof. Michael Stallcup and Prof.
Woojin An. They brought me some novel ideas about my research. They also shared their
research experiences that may help in my research. Also, they helped me with my future
career as well.
Then, I would like to thank Dr. Baruch Frenkel and Prof. Pragna Patel. Besides teaching
us knowledge in the courses, they also gave me a lot of advices throughout my research as
well as helping me prepare the oral interview.
Next, I want to thank Dr. Sonya Williams. She was a member of our lab and my project is
actually the extension of her works. She taught me the way this lab does the experiments.
We communicated a lot when she was still in the lab about the experiment plans. And she
also helped with my part of work when I had to go for the coursework or couldn’t get to
health science campus so early. She left many beneficial experiences that made my study
much easier.
I also want to thank members of Dr. Rice’s lab: Vivian Chen and Benjamin Weekley, and
members of Dr. An’s lab. They helped me a lot with my experiment and also discussed
with me about the problems I met. Working as a team really help to overcome problems
and avoid severe mistake.
Finally, I want to say thank you to my parents, who grew me up as a researcher and
support me financially and spiritually throughout my life.

iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS .............................................................................................................................. ii
List of Table .......................................................................................................................................................... v
List of Figures ...................................................................................................................................................... v
Abstract ................................................................................................................................................................. vi
Chapter 1: Introduction ....................................................................................................................................1
Nucleosomes, histones and histone modifications ...........................................................................1
Histone tail cleavage .................................................................................................................................2
Histone protease:........................................................................................................................................3
Strategies to discover H3NT proteases ...............................................................................................5
Rationale and Research Goals ...............................................................................................................6
Chapter 2: Results ........................................................................................................................................... 11
Developing an H3 N-terminal tail (H3NT) substrate: Rationale and Approach ............... 11
....................................................................................................................................................................... 12
The rHis-H3NT protein is a useful, but not optimal, substrate for H3NT zymography 13
Generating a larger rH3NT substrate: rHis-2xH3NT................................................................ 15
Generating a larger rH3NT substrate: crosslinking rHis-H3NT ........................................... 15
pET45-5xHis-H3NT-Cys: robust expression but no dimer formation ................................. 16
pOP2H-Cys-H3NT-8xHis: dimer formation in bacteria but low yield ................................ 20
pET23a-Cys-H3NT-5xHis: low expression .................................................................................... 20
Determining the utility and limitations of the rH3NT-His substrate in the in vitro H3
iv

cleavage assay ........................................................................................................................................... 21
Chapter 3: Discussion ............................................................................................................................... 25
Chapter 4: Materials and Methods ...................................................................................................... 28
Plasmid construction: ............................................................................................................................ 28
Peptide induction and purification: .................................................................................................. 28
Crosslinking .............................................................................................................................................. 28
Western blot: ............................................................................................................................................ 29
Zymography: ............................................................................................................................................ 29
In vitro proteolysis: ................................................................................................................................ 29
Bibliography .................................................................................................................................................... 30

v

List of Table
Table 1 ........................................................................................................................... 8

List of Figures
Figure 1 ......................................................................................................................... 9
Figure 2 ....................................................................................................................... 10
Figure 3 ....................................................................................................................... 12
Figure 4 ....................................................................................................................... 14
Figure 5 ....................................................................................................................... 19
Figure 6 ....................................................................................................................... 24
vi

Abstract

Proteolysis of the histone H3 N-terminal tail (H3NT) within chromatin is a frequent and
conserved epigenetic event in eukaryotes. The proteases responsible for H3NT “clipping” remain
largely unknown, which has hampered advances in the field to understand the biological
significance of H3NT “clipping”. The goal of my thesis research was to develop an H3NT
substrate that could be used in a variety of standard in vitro approaches to greatly accelerate the
discovery and investigation of H3NT proteases. Using a bacterial expression system, I generated
and purified robust amounts of a recombinant protein that contains 17 tandem repeats of the
histone H3 N-terminal tail (rH3NT). My studies validate the utility of the rH3NT substrate in the
in vitro H3 cleavage assay and zymography approaches to detect H3NT protease activity using
the positive controls, MMP-2 and MMP-9. However, my results also demonstrate that the rH3NT
substrate requires refinements to increase the sensitivity of H3NT protease activity detection in
the H3 cleavage assay and to improve substrate immobilization for zymography. These
forthcoming refinements and considerations are discussed.

1

Chapter 1: Introduction

Nucleosomes, histones and histone modifications
In eukaryotic cells, the fundamental unit of chromatin is nucleosome, which is composed
of a histone octamer with two copies of H2A, H2B, H3, and H4 and 147bp DNA chain wrapping
around it. The histone-histone or histone-DNA interaction largely depends on histone C-terminal
domain. All 4 histone proteins have a similar histone fold motif consists of 2 short α-helices and
one relatively long α-helix linked by β-loops on C-terminus that will interact with each other in a
“handshake” model. [1] Histone-DNA interaction is achieved by the insertion of C-terminal
arginines into DNA minor groove. Because of this, C-terminus of histone proteins are usually
wrapped in the core of nucleosome. The exception is H2A. The sensitivity of its C-terminus to
proteases indicates that the C-terminus of H2A is exposed outside. Each of the histone proteins
has a structurally undefined N-terminal tail rich in alkaline amino acids. The N-terminal tail
contributes to the stability of nucleosome and the interaction between nucleosomes in chromatin
fiber. The removal or some modification of histone N-terminal tail is efficient to influence the
nucleosome stability. [2] The Histone tail can be post-translationally modified in several ways
and the posttranslational modifications (PTMs) of histone proteins can be removed by reverse
reaction or nucleosome turnover. Lysine on histone tail can be acetylated by histone
acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs). Usually
acetylation of histone tail neutralizes a positive charge on histone tail, making histone octamer
2

and DNA more likely to dissociate, so that the nucleosome is unwrapped and the chromatin
domain becomes more accessible. [3] As a result, the transcription machinery can recruit at the
histone-acetylated promoter easier and initiate transcription more frequently. Histone serines,
threonines and tyrosines can be phosphorylated by kinases and dephosphorylated by phosphatases.
Phosphorylation of histone protein can act in the similar way as acetylation, and it can also create
specific site for protein recognition modules binding. [4] Histone methylation usually happens on
lysine or arginine by corresponding methyltransferase (HMT) and demethylation is caused by
demethylase. Arginine can be mono-methylated or di-methylated while lysine can even be
tri-methylated. Methylation doesn’t change the charge of histone tail, so it functions in a more
specific way. [5] H3K4, H3K9, and H3K27 are frequent methylated sites. H3K4me usually marks
active enhancer, while H3K4me3 happens mostly at active promoter. Methylation at H3K9
usually indicates gene repression or heterochromatin. H3K27me is a repression marker while
H3K27me3 serves as a flexible regulator of dynamically regulated genes. PTMs can alter the
nucleosome stability by influencing the interaction between histone and linker DNA or other
histone proteins, or by influencing the chromatin binding factor, thus regulating the gene
expression [6].

Histone tail cleavage
Histone tail cleavage is a kind of irreversible histone PTM. Truncated histone protein was first
observed in 1976, when Eickbush et al. described a C-terminal clipped H2A species discovered in
3

calf thymus chromatin [7]. Histone tail cleavage has been reported in various species from yeast
to human, and during various processes from cell differentiation to virus replication. Just like
other histone PTMs, histone tail cleavage has been observed in all four histone proteins but
mostly in histone H3 N-terminal tail (H3NT) [8]. Histone tail cleavage can influence gene
expression in several ways. Firstly, the deletion of histone tail will directly influence nucleosome
stability. [9] Secondly, the deletion of histone tail will remove other histone modifications on the
tail, such as H3K4me, H3S10ph, etc. thus causing similar effect as deacetylation or
dephosphorylation. Finally, histone tail clipping may also be a promoter of nucleosome turnover.
[10]

Histone protease:
Several different proteases are involved in histone tail clipping. Histone protease may have one
or multiple cleavage sites, some of them even have cut sites on different histone proteins [11].
Certain cell differentiation event usually involves specific histone protease. Histone H3
N-terminal tail clipping has been observed in 1984 in foot-and-mouth-disease-virus infected cell
[12,13]. This cleavage event is identified to be caused by FMDV protease 3C and it’s believed to
be related to virus-induced host cell transcription shutoff. In 2008, a H3NT clipping event is
described in S. cerevisiae during stationary phase and sporulation [10]. This endopeptidase was
identified as a kind of serine protease and it may help activating sporulation and stationary-phase
induced genes by removing inhibitory modification on H3NT. The cleavage event was then
4

proved to be related to Prb1 [14]. As the in vitro cleavage site of Prb1 is K23 while the cleavage
event in vivo happens at T21, it’s not settled whether the cleavage is caused by Prb1 directly but
the K23 activity is inhibited in vivo or by Prb1 activating another histone protease. In
Plasmodium falciparum, a H3NT cleavage event was observed during intra‐erythrocytic stages
[15]. The histone protease responsible for this clipping is a Cathepsin-C like protease (most likely
to be DPAP). This H3NT cleavage event targets 12 genes, 6 of which involve in DNA replication
and repair (RPA1, PCNA, SSB, TOPO‐I, TOPO‐II, DNA pol α), and it always begins within the
5′UTR and spans a region of approximately 1.5 kb. More H3NT proteases were discovered in
vertebrates. Cathepsin L has been well identified as a H3NT protease that cleaves at H3A21 and
then chews back till H3K27 [16]. Its targets are cell cycle regulating genes such as NUF2,
AURKB, RRM2, KI67, PLK1, and SASP related genes such as TGFB2, and IGFBP7 and it has
been proved to play an important role in embryo stem cell differentiation and senescence [17].
Cathepsin D is found to cleave H3K23 in mouse mammary gland, which is believed to be related
to post-lactational regression [18]. Tryptase can cleave both H2B N-terminal tail and H3NT [19],
but most of the current functional studies focus on H2B. MMP-9 will enter the nuclei and cleave
H3K18 during osteoclast precursor cell differentiation [20, 21]. It may lead to H3 displacement at
osteogenesis related genes like Nfatc1, Lif, and Xpr1 and induce the expression, thus regulating
osteoclast differentiation. JMJD5 is also reported to be able to cleave H3 [22]. It can cleave
H3K9 as a Cathepsin L-like protease in vivo, while core-JMJD5 also shows H3R2~R7 peptidase
activity in vitro [23]. JMJD5 can suppress genes with H3K4/K9me1 such as stat1 and stat4, so it
5

is predicted to have the ability to regulate cell cycle or growth/survival. Glutamate
Dehydrogenase [24] and Legumain [25] are also reported to have the ability to cleave H3NT at
specific site in vitro, but they are not proved to cause tail clipping in vivo. These findings support
a hypothetical model where different proteases are utilized by the cell as an H3NT protease in a
differentiation-dependent context. Interestingly, many of these histone proteases were not even
considered as nucleoprotein before deep researches were done. Although truncated histone H3
was frequently observed, it’s quite effort-taking to make a causal connection between a cleavage
event and a certain enzyme. As a result, the nuclear proteases responsible for H3NT clipping
remain largely unknown.

Strategies to discover H3NT proteases
The traditional way to discover chromatin-modifying enzymes is to purify soluble nuclear
extract by chromatography and then perform in vitro activity assays on every fraction. The
fraction displaying the highest enzyme activity is isolated for mass spectrometry and/or inhibitor
assays to identify the range of possible enzymes. The success of these assays is dependent on the
quality and sensitivity of an appropriate substrate.
But inhibitor assay requires firstly known enzyme working environment for in vitro
proteolysis test, secondly a complete understanding of species genome so that after narrowing the
enzyme to a specific class, we could do knockdown assay to precisely make sure the enzyme is
6

responsible. And if there are more than one protease or the protease needs interaction with other
protein to keep active, additional difficulty will be added to identify the specific protease.
It is lucky that some of the histone proteases, such as Cathepsin L and MMP-9, can also
degrade collagen. So, gelatin zymography also played an important role in detecting enzyme
activity in those researches. Zymography is a method the peptide substrate was added into the
SDS-PAGE gel mixture before it’s set. After running the gel, the protease is separated from other
components and moved to a specific place in the gel. Then the gel is incubated in Triton to
remove SDS. The protease will be able to renature and partially recover activity. Finally, the gel
is incubated in developmental buffer to allow the enzyme to chew the substrate in the gel and the
gel is stained by Coomassie blue. The substrate will be stained blue but the protease will chew a
blank zone without substrate. The benefit of this method is firstly it doesn’t require a large
amount of sample. Secondly it can both detect/quantitate the enzyme and separate the enzyme.
However, traditional gelatin zymography of course can only detect gelatin proteases. Not all
histone proteases can cleave gelatin, nor all the proteases that can cleave gelatin are histone
proteases. The substrate used frequently in in vitro proteolysis assay: core histone protein is too
small to be used as zymography substrate. In fact, even if it’s large enough, as large part of
histone remains uncut when the tail is clipped, it can still not be a zymography substrate.

Rationale and Research Goals
7

As illustrated in Figure 2, the goal of my thesis research was to create and validate a
recombinant H3NT protein (rH3NT) substrate that could be utilized in different in vitro assays to
discover and characterize novel H3NT proteases. Ideally, the rH3NT substrate would first be used
in zymography as a 1-step approach to identify and discover H3NT proteases present in nuclear
extracts. The major technical requirements for zymography include the need for robust amounts
of the rH3NT protein (1 mg/ml final) and the substrate must be large enough to be immobilized in
the gel. Following discovery of the putative H3NT protease by zymography, the rH3NT substrate
would be used to validate and characterize H3NT activity of the protease using the in vitro H3
cleavage assay. The utility of this assay is dependent on the sensitivity of the rH3NT substrate to
detect limiting amounts of H3NT protease activity within biological samples, such as nuclear
extracts. If the rH3NT substrate displays high sensitivity for H3NT protease activity, a modified
version of the substrate would then be generated to accommodate quantitative high throughput
functional assays to characterize H3NT protease activity and to rapidly screen for H3NT
proteases in other biological samples.

8

Name Type Cut site Cell
line/Tissue
Predictied
function
MMP-9 Metalloproteinase K18 Mouse
primary
osteoclast
precursor cell
osteoclastogenic
gene activation
Prb1 (proteinase B) serine protease Lys23(in vitro) Yeast

Yeast
endopeptidase(likely
to be Prb1)
serine protease T21 Yeast cause H3
displacement
and induce gene
expression
Cathepsin-C like
protease (likely to be
DPAP)
serine cysteine
protease
A21 Plasmodium
falciparum
intra ‐
erythrocytic
stages
coordinate
DNA synthesis
and subsequent
karyokinesis
JMJD5 (KDM8) demethylase/
hydroxylase/
CTSL like
protease
K9 HeLa/A549 regulate cell
cycle or
growth/survival
FMDV 3C protease

Leu21 picornaviridae
infected
BHK21
host cell
transcription
shutoff
Cathepsin D aspartic protease K23 Mouse
mammary
gland
post-lactational
regression
Cathepsin L cysteine protease A21(primary)~K27(final) ESC, IMR90
fibroblast,
Melanocytes
ESC
differentiation,
induce
senescence
Table 1. Known H3 N-terminal tail proteases

9

Fig 1. Nucleosome structure and H3 N-terminal tail cleave sites

10

Figure 2. The goal of my MS thesis project is to create and validate an H3NT peptide substrate that
can be used in multiple applications to discover and characterize novel H3NT proteases. First, the
substrate will be used in zymography as a 1-step approach to identify and discover H3NT proteases
present in nuclear extracts. Next, the substrate will be used to validate and characterize H3NT
activity of the protease in in vitro and gel-based assays. After these assays confirm the utility of the
H3NT substrate, the peptide will be modified to accommodate high throughput functional screening
assays.

11

Chapter 2: Results

Developing an H3 N-terminal tail (H3NT) substrate: Rationale and Approach
The overall goal of my thesis project was to develop a recombinant H3NT (rH3NT) peptide
substrate to discover and investigate H3NT proteases using standard in vitro approaches. To this
end, a synthetic cDNA containing 17 tandem repeats of histone H3 amino acid sequences
A7~S28 was generated using standard gene synthesis (Genewiz). This sequence includes most of
the currently known H3NT proteolytic sites, as illustrated in Figure 3. The H3NT cDNA was then
subcloned by Gibson assembly into the pET45 bacterial expression vector, which contains a
5xHis-tag at the 5’ end of multiple cloning site (Fig 4A). Following Sanger sequencing
verification, the pET45-H3NT plasmid was transformed into the BL21-derived Rosetta bacteria
line for expression and purification of the rHis-H3NT protein. Although the protein was rapidly
and robustly expressed after induction with 1 M IPTG, rHis-H3NT was enriched within inclusion
bodies rather than the soluble fraction. This required the purification and isolation of rHis-H3NT
under denaturing conditions, as described in Materials and Methods. Following purification of
rHis-H3NT by Ni-NTA affinity chromatography, the protein was dialyzed into water. This
approach resulted in robust yields of rHis-H3NT, as high as 2 mg/ml (Fig 4B). To validate the
utility of the rHis-H3NT as a viable substrate for H3NT proteases, an in vitro H3 cleavage assay
was performed using the known H3NT protease, MMP-9. As shown in Figure 4C, recombinant
MMP-9 (rMMP9) readily proteolyzes the histone H3 positive control as well as the rHis-H3NT
12

substrate. These results demonstrate that the rHis-H3NT can be used as a substrate to detect and
investigate H3NT protease activity in in vitro assays.

Figure 3. Creation and purification of the recombinant H3NT substrate. (Top) Standard gene
synthesis was used to generate the H3NT substrate, which contains 17 repeats (blue bars) of histone
H3 amino acid sequence 7-28. The sequence includes all of the H3NT clipping sites (red) currently
known. (Bottom) The sequence was subcloned into the pET45 His-tagged vector and transformed into
BL21 E. coli. His-rH3NT expression was induced in log phase cells treated with 1 mM IPTG for 8
hours at 30C. Since rH3NT is insoluble, cells were denatured in 8M urea prior to Ni-NTA purification
using the manufacturer’s protocol (Qiagen). Coomassie staining of the SDS-PAGE demonstrates
abundant His-rH3NT in elutions 1 and 2, which were combined and dialyzed in water.

13

The rHis-H3NT protein is a useful, but not optimal, substrate for H3NT zymography
The next goal of my thesis project was to leverage the rHis-H3NT protein as a substrate in
zymography to discover H3NT proteases. To test this, a 12% SDS-PAGE was prepared with 1
mg/ml (final) of the rHis-H3NT substrate or gelatin, the positive control. The gel lanes were
loaded with decreasing amounts of rMMP9 prior to electrophoresis. As shown in Figure 4D,
robust proteolysis of the gelatin positive control was detected even at the lowest concentrations of
rMMP9. Although rMMP9 protease activity was also detected with the rHis-H3NT substrate, the
sensitivity of protease activity detection was dramatically lower compared to gelatin. Consistent
with this, MMP-2 H3NT protease activity present in nuclear extracts of differentiating C2C12
cells (unpublished) was clearly detected by gelatin zymography but not detected by zymography
using the rHis-H3NT substrate (data not shown). While these results demonstrate the feasibility
of the proposed H3NT zymography approach to discover novel H3NT proteases, the results also
indicate that rHis-H3NT is a sub-optimal substrate for zymography.

14

Figure 4. Expression and purification of rHis-H3NT substrate. (A) Schematic of the
pET45-5xHis-H3NT plasmid. (B) SDS-PAGE of Ni-NTA eluted fractions from pET45-5xHis-H3NT
induced bacteria. (C) SDS-PAGE of an in vitro H3 cleavage assay using core histone substrates or
serial dilutions of the rHis-H3NT substrate in the presence or absence of rMMP9 protease. (D)
Gelatin zymograpy (top) or rHis-H3NT zymography (bottom) using serial dilutions (triangle) of
rMMP9 protease. (E) Schematic of the pET45-5xHis-2xH3NT plasmid. (F) SDS-PAGE of lysates
from uninduced or induced pET45-5xHis-2xH3NT bacteria.

15

Generating a larger rH3NT substrate: rHis-2xH3NT
The most likely reason why rHis-H3NT does not perform well in zymography is because of
the relatively small size of the substrate. In contrast to gelatin (>100 kDa), the smaller
rHis-H3NT (~35 kDa) is not effectively polymerized in the gel and migrates out of the gel during
electrophoresis. This is clearly evident by the absence of the substrate at the top of the gel and the
accumulation of rHis-H3NT at the bottom of the gel (Figure 4D). Therefore, we reasoned that
generating a larger rHis-H3NT substrate would overcome this technical barrier. Using Gibson
assembly, I successfully inserted a second copy of the H3NT cDNA into the pET45-5xHis-H3NT
plasmid (Figure 4E). The pET45-5xHis-2xH3NT plasmid was transformed into Rosetta bacteria
cells and induced using 1 M IPTG, as previously described. Despite several attempts and changes
to induction conditions, expression of the rHis-2xH3NT was never detected at sufficient
concentrations for use in zymography (Figure 4F). The failure to express the >70 kDa
rHis-2xH3NT is likely due to the technical limitations of bacteria to effectively express large
proteins. Regardless, these negative results indicate that alternative strategies to generate a large
H3NT substrate for zymography were necessary.

Generating a larger rH3NT substrate: crosslinking rHis-H3NT
Chemical crosslinking approaches were examined as a means to leverage the robust yields of
rHis-H3NT to generate a larger H3NT substrate suitable for zymography. We first considered
crosslinking rHis-H3NT to a large carrier protein to increase the overall size and improve
16

immobilization of the substrate in the gel. However, we reasoned that addition of this large
non-specific carrier protein could yield false positive or false negative results and, therefore,
confound accurate detection of H3NT proteases. In contrast to crosslinking to a large non-specific
carrier protein, we reasoned that crosslinking multiple molecules of rHis-H3NT to themselves
would not only increase the size of the substrate but also the specificity and sensitivity of the
substrate for zymography. Chemical crosslinking of lysines, which are abundant in the H3NT,
was first considered as a convenient strategy but there are two reasons why this approach was not
pursued. First, arbitrary crosslinking of lysines would result in a multitude of unpredictable
H3NT substrates that may confound meaningful interpretation of the subsequent zymography
results. Secondly, crosslinking of lysines could lead to the physical occlusion of the protease from
functioning at its consensus sequence, resulting in a false negative. For example, MMP-9 cleaves
the H3NT at K18 [19] and, therefore, the crosslinking of K18 to another lysine would likely
prevent MMP-9 from proteolyzing this substrate. As an alternative strategy to the arbitrary
crosslinking of lysine residues, the crosslinking of cysteine residues, via a free thiol group, could
provide a means to generate a single large rH3NT substrate. Since the rHis-H3NT substrate lacks
cysteine residues, we reasoned that inserting a single cysteine within the coding region would
allow the generation of an ~70 kDa rH3NT dimer via covalent thiol linkage; a substrate that
should be suitable for zymography.

pET45-5xHis-H3NT-Cys: robust expression but no dimer formation
17

Gibson assembly was used to add a cysteine on the C-terminus to generate the
pET45-5xHis-H3NT-Cys plasmid (Figure 5A), as described in Materials and Methods. The
plasmid was transformed into the Rosetta bacteria cells and induced to express the
rHis-H3NT-Cys protein, as described above. The pET45 plasmid consistently resulted in the
robust expression and purification of rHis-H3NT-Cys (Figure 5B). Following Ni-NTA
purification, 2 higher molecular weight bands were detected by SDS-PAGE suggesting that the
rHis-H3NT-Cys was naturally generating disulfide bonded dimers within the bacteria. Consistent
with this, treating the samples with DTT to reduce disulfide bonds resulted in the ablation of
these bands (Figure 5B; first lane vs. second lane).
For crosslinking of rHis-H3NT-Cys, the purified protein was dialyzed into conjugation buffer.
Increasing concentrations of the BM(PEG)2 crosslinker [26] in DMSO (5-20 mM final) were
applied to the samples to crosslink cysteine residues, and the reaction is quenched by DTT prior
to SDS-PAGE analysis. Unexpectedly, rHis-H3NT-Cys dimer formation was not detected in any
of the samples (Figure 5B). Similar negative results were obtained using alternative thiol
bond-promoting strategies including air oxidation [27] (not shown), DMSO oxidation [28] and
H2O2 oxidation [29] (Figure 5C). One likely explanation for these negative results is that the full
length rHis-H3NT-Cys protein is not efficiently translated in bacteria and, therefore, the
C-terminal cysteine is not present for crosslinking. Consistent with this, SDS-PAGE following
Ni-NTA purification of the pET45 N-terminal 5xHis tag illustrates the predominance of smaller
proteins that likely represent premature termination of rHis-H3NT-Cys translation (Figure 4B).
18

Therefore, despite robust expression of rHis-H3NT-Cys using the pET45 plasmid, the results
indicate that an alternative approach to N-terminal tag purification was necessary for generation
of the full-length recombinant protein.
19

Figure 5. Purification and crosslinking of rH3NT-Cys. (A) Schematic of the pET45-5xHis-H3NT-
Cys plasmid. (B) SDS-PAGE of Ni-NTA purified pET45 rHis-H3NT-Cys protein monomer and dimer
(input; arrows). Crosslinked dimer was not observed after incubation with increasing concentrations
of the BM(PEG) 2 crosslinker (triangle), or with H 2O 2 or DMSO (C). (D) Schematic of the
pOP2H-Cys-H3NT-8xHis plasmid. (E) SDS-PAGE of Ni-NTA purified pOP2H rCys-H3NT-His
dimer (arrow). (F) Western analysis of pOP2H-Cys-H3NT-8xHis lysate and Ni-NTA purified fraction
using an anti-His antibody. (G) Schematic of the pET23a-Cys-H3NT-5xHis plasmid. (H) SDS-PAGE
of pET23a-Cys-H3NT-5xHis uninduced and induced cell lysates.
20

pOP2H-Cys-H3NT-8xHis: dimer formation in bacteria but low yield
We reasoned that a bacterial expression plasmid containing a C-terminal affinity tag would
ensure the purification of full length rH3NT. Gibson assembly was used to clone an N-terminal
cysteine-H3NT insert into the pOP2H bacterial expression vector, which contains a C-terminal
8xHis tag (Figure 5D) [30]. The pOP2H-Cys-H3NT-8xHis plasmid was transformed in BL21
bacteria cells and induced with 1 M IPTG, as described above. SDS-PAGE following Ni-NTA
purification revealed that the predominant purified protein was ~70 kDa, consistent with dimer
formation of rCys-H3NT-His in bacteria (Figure 5E). To test this hypothesis, Western analysis
using an anti-His antibody was performed. The results confirmed that the predominant band is
dimerized rCys-H3NT-His but also revealed the presence of ~19 other rCys-H3NT-His dimers
not detected in the Coomassie-stained SDS-PAGE (Figure 5F). Based on the pET45-H3NT
results, we speculate that these bands represent prematurely terminated forms of rCys-H3NT that
retain the capacity to dimerize to full length rCys-H3NT-His in bacteria, due to the N-terminal
cysteine. Regardless of the heterogeneity, the larger size of these rCys-H3NT-His dimers may
improve immobilization of the rH3NT substrate in SDS-PAGE for zymography. However, since
zymography requires high amounts of substrate (~10 mg/gel), the consistently low yields of
rCys-H3NT-His protein preclude the utility of the pOP2H-Cys-H3NT-8xHis system for H3NT
zymography.

pET23a-Cys-H3NT-5xHis: low expression
21

In contrast to pOP2H, the pET expression system produces robust yields of rHis-H3NT (Figure
4A). Therefore, Gibson assembly was used to clone an H3NT or Cys-H3NT insert into the
pET23a bacterial expression vector, which contains a C-terminal 5xHis tag (Figure G). The
plasmids were transformed into Rosetta bacteria cells and induced with 1 M IPTG, as described
above. As expected, Ni-NTA purification of pET23a-H3NT-His expressing cells resulted in
robust yields of rH3NT-His (Figure 6A,B). Surprisingly, induction of pET23a-Cys-H3NT-His
cells failed to generate any detectable amounts of rCys-H3NT-His protein in SDS-PAGE (Figure
5H). It remains unclear why the addition of an N-terminal cysteine prevents expression of full
length rCys-H3NT-His in the pET23a system, especially since the identical insert in the pOP2H
system did generate detectable amounts of rCys-H3NT-His (Figure 5E). While troubleshooting
this problem the laboratory was unexpectedly closed due to the coronavirus pandemic. My
strategies to overcome this technical barrier are described in the Discussion.

Determining the utility and limitations of the rH3NT-His substrate in the in vitro H3
cleavage assay
Although the H3NT substrate has not yet been optimized for use in zymography, the
rH3NT-His protein was evaluated for its utility as an H3NT protease substrate in established in
vitro H3 cleavage assays. First, the pET23a-H3NT-5xHis expression system was used to generate
robust yields of purified full length rH3NT-His protein (Figure 6A, B). Next, conditioned media
and nuclear extracts from differentiating C2C12 cells were collected, as described in Materials
22

and Methods. Differentiating C2C12 cells express and secrete high levels of MMP-2, an H3NT
protease (unpublished), into the media, as well as transporting relatively smaller amount of
MMP-2 to the nucleus. Our goal was to leverage these MMP-2-rich biologically relevant samples
to evaluate the utility of the rH3NT-His protein as a substrate for MMP-2-mediated proteolysis.
Gelatin zymography was first performed to confirm MMP-2 activity in the C2C12 conditioned
media and nuclear extracts (Figure 6C). Next, the in vitro H3 cleavage assay was performed using
the rH3NT-His substrate and the MMP-2-rich conditioned media under varying experimental
conditions. The results indicate, as expected, that the proficiency of rH3NT-His substrate
proteolysis was directly dependent on the amount MMP-2 activity in the conditioned media and
incubation time (data not shown). These optimized experimental conditions were then used to
evaluate the C2C12 MMP-2-containing nuclear extracts to proteolyze the rH3NT-His substrate.
In contrast to the C2C12 conditioned media and rMMP-2 positive control, the C2C12 nuclear
extracts failed to proteolyze the rH3NT-His substrate in vitro (Figure 6D). One possible technical
reason for this negative outcome may be due to the high NaCl concentration (400 mM) of the
nuclear extracts compared to the DMEM media (110 mM), which may have an inhibitory effect
on MMP-2 H3NT protease activity. To test this, the in vitro H3 cleavage assay was repeated with
the C2C12 MMP-2-rich conditioned media under varying NaCl concentrations and significantly
shorter incubation time (2 hours instead of 24 hours). Consistent with the hypothesis, the results
indicate that NaCl concentrations >55 mM have a progressively inhibitory effect on MMP-2
H3NT protease activity in vitro (Figure 6E). Based on these findings, the in vitro H3 cleavage
23

assay was repeated with C2C12 nuclear extracts that had been dialyzed in water or DMEM in
order to exclude the observed NaCl-associated inhibition of MMP-2 activity. In both cases,
however, the C2C12 MMP-2-containing nuclear extracts failed to effectively proteolyze the
rHis-H3NT substrate in vitro (data not shown). Although these data demonstrate the utility of the
rHis-H3NT substrate to investigate H3NT protease activity of recombinant enzymes or highly
active biological samples in vitro, this visual SDS-PAGE approach currently lacks the sensitivity
necessary to detect H3NT protease activity in nuclear extracts.

24

Figure 6. Evaluation of the rH3NT-His substrate in in vitro assays. (A) Schematic of the pET23a-
H3NT-5xHis plasmid. (B) SDS-PAGE of Ni-NTA purified rH3NT-His protein. (C) Gelatin
zymography using decreasing amounts (triangle) of differentiating C2C12 MMP-2-rich conditioned
media or C2C12 soluble nuclear extracts. (D) SDS-PAGE following in vitro H3 cleavage assay with
the rH3NT-His substrate in the presence or absence (+/-) of rMMP2 or C2C12 soluble nuclear
extracts. (E) SDS-PAGE following in vitro H3 cleavage assay performed with varying amounts of
C2C12 MMP-2-rich conditioned media and NaCl concentrations, as indicated.
25

Chapter 3: Discussion

The overall goal of my thesis project was to develop a recombinant H3NT (rH3NT) protein
substrate for use in a variety of standard in vitro approaches to greatly accelerate the discovery
and investigation of H3NT proteases. By creating an expression construct containing 17 tandem
copies of the H3NT (aa7-28/copy), I was able to successfully generate robust amounts of a
rH3NT-His protein using a bacterial expression system. My findings validate the utility of
rH3NT-His as a substrate in the in vitro H3 cleavage assay using the H3NT proteases, MMP-2
and MMP-9, or MMP-2-rich media collected from differentiating C2C12 cells. These results
indicate that the rH3NT-His can be used to investigate H3NT protease activity of recombinant
enzymes or highly active biological samples in vitro. However, my results also demonstrate that
the rH3NT-His substrate fails to detect, the relatively smaller amounts of, MMP-2 H3NT protease
activity present in nuclear extracts isolated from differentiating C2C12 cells. These results
demonstrate the low sensitivity and limitations of rH3NT-His as a substrate in the in vitro H3
cleavage assay to discover limiting H3NT protease activity in biological samples.
As an alternative in vitro method to discover H3NT proteases, I adopted a zymography
approach using the rH3NT-His substrate and successfully detected rMMP-9 activity. Similar to
above, I was not able to detect H3NT protease activity in the C2C12 nuclear extracts using the
H3NT zymography approach. However, my data clearly indicate that the likely reason for this
negative outcome is that the rH3NT-His substrate is too small to be effectively immobilized in
26

the gel and, therefore, is not suitable for zymography in its current form. To overcome this
technical barrier, I attempted to generate a larger rH3NT-His substrate using the same bacterial
expression system. However, the limitations of this bacterial system to generate robust amounts
of a large recombinant protein precluded generation of the rH3NT-2x substrate. As an alternative,
I planned to leverage a thiol crosslinking approach to covalently bind the smaller purified
rH3NT-His proteins together, thus generating a large H3NT substrate suitable for zymography.
This should have been straightforward but I encountered several unexpected outcomes and
difficulties, as detailed in Results. Before the lab closed due to the coronavirus pandemic, I was
working on expressing a pET23a-H3NT-Cys-5xHis construct (C-terminal cysteine) for
subsequent thiol crosslinking. Although this has yet to be optimized, the robust expression of
recombinant proteins in the pET expression system and the well-documented effectiveness of
protein-protein crosslinking suggest that this approach will yield a suitable rH3NT substrate large
enough for use in zymography.
My results demonstrate that the rH3NT-His protein can be used as a substrate in the in vitro
H3 cleavage assay and zymography to detect MMP-2 and MMP-9 H3NT protease activity.
However, my data also demonstrate that the sensitivity of the rH3NT-His substrate to detect
protease activity is much less compared to the optimal MMP-2/9 substrate, gelatin. These
findings illustrate the possible limitations and caveats of the rH3NT substrate, in its current form,
to discover and investigate H3NT proteases. Bacterially expressed recombinant proteins lack
post-translational modifications, which may result in decreased H3NT protease activity for the
27

substrate. For example, MMP-9 H3NT protease activity is significantly increased when its target
residue, K18, is acetylated compared to an unmodified substrate. Therefore, the absence of
acetylation in the rH3NT substrate likely accounts for the decreased detection of MMP-2/9 H3NT
protease activity observed in the in vitro H3 cleavage assay and zymography. It remains unknown
whether the activity of H3NT proteases other than MMP-2/9, such as the cathepsins, are also
directly affected by H3 post-translational modifications but this is an important consideration
when investigating H3NT protease activity using an unmodified substrate alone. Another
important consideration is that several histone-modifying enzymes display exclusive, or
significantly higher, activity for nucleosomal substrates compared to peptide or recombinant
protein substrates. However, this consideration is likely less applicable to H3NT proteases, such
as the MMPs and cathepsins, which have been well-documented to display robust activity using
short peptide substrates. My collective results support the utility of a rH3NT protein substrate to
discover and investigate H3NT proteases but my results also demonstrate that the current rH3NT
is a suboptimal substrate that requires significant refinements to achieve meaningful experimental
outcomes.

28

Chapter 4: Materials and Methods
Plasmid construction:
rH3NT peptide sequence was ordered and synthetized from Genewiz. The original sequence is
in pUC57 vector and we amplified the sequence by PCR with a pair of primers that have 30 bp
overlap with pET45 vector. The fragment was purified by ZR Plasmid Miniprep kit and then
inserted into BamH1 cut pET45 vector by Gibson Assembly using NEB Gibson Assembly
®
kit.
The assembled product was transformed into DH5α bacteria and planted on Amp
+
plates. The
monoclonal colonies were then picked and incubated in 37℃ overnight. The recombinant
plasmid was purified by ZR Plasmid Miniprep kit. After grossly identified by agarose
electrophoresis, the plasmid was sent to IDT
®
for sequencing.

Peptide induction and purification:
The recombinant plasmid was transformed into BL21 bacteria. Incubate until the OD600
reached 0.8. Add IPTG at a final concentration of 1mg/ml. Shake the bacteria overnight. The
bacteria were spun down and resuspended by pH 8.0 QB+ buffer (8M Urea, 0.1M NaH2PO4,
10mM Tris base, 0.5M NaCl, 10% glycerol, 1% Triton X-100, adjust pH by HCl) and lysed by
sonication under 4℃. The mixture was centrifuged under 20000g for 15min to remove the
insoluble fragments and then incubated with QIAGEN Ni-NTA Agarose beads for 1 hour on
shaker at room temperature. The beads were washed by pH 6.0 QB+ buffer and the protein were
eluted by pH 4.0 QB+ buffer. For small scale purification, 20μl of balanced beads were washed
by 200μl of buffer twice and eluted by 60μl of elution buffer. For large scale purification/
concentration, 1ml of beads were washed by 10ml of wash buffer, then resuspended by another
10ml of wash buffer and loaded on the column. After wash buffer flew through, add 6ml of
elution buffer and collect 500μl of fraction in each microtube. The unbound material from the
first-round beads binding were incubated with the beads again and went for a second-round
elution.
After running the 12% SDS-PAGE gel under 120V and Coomassie blue staining, the fractions
containing target protein were mixed and dialyzed in 6M urea, 4M urea, 2M urea and finally
water using 12000~14000MW tubin. Each dialyze step last for at least 1 hour.

Crosslinking
BM(PEG)2: 3.1mg of BM(PEG)2 was dissolved in 0.5ml of DMSO. The substrate was dissolved
into pH7.2 PBS with 5mM EDTA. 10μl of crosslinker solution was added into 1ml of substrate.
The mixture was incubated at room temperature for 1 hour and the reaction was quenched by
10mM DTT.

DMSO/ H2O2: 10%~30% DMSO or 5%~15% H2O2 was added into 1ml the substrate solution to
reach a final volume of 1ml. The mixture was incubated at room temperature for 1 hour.

Air oxidation: 1ml of substrate solution was incubated at 4℃ overnight with lid opened.
29

Western blot:
Samples were subjected to 12% SDS-PAGE gel and then transferred to PVDF membranes and
hybridized by 1:1000 His-tag rabbit antibody overnight. Then the protein was visualized by
1:20000 HRP-conjugated goat anti-rabbit secondary antibody.

Zymography:
Substrate was added into the 12% SDS-PAGE gel at a final concentration of 1mg/ml. After
electrophoresis, the gel was washed in 2.5% Triton X-100 for 30 min twice. The gel was washed
in developing buffer (50mM Tris base, 10mM CaCl2, 0.02%NaN3) once and then incubated in
developing buffer at 37℃ overnight. The gel was stained by Coomassie blue.

In vitro proteolysis:
10μl of sample was mixed with 5μl of substrate and 5μl of 4xH3Cl buffer (200mM Tris base,
40mM CaCl2, 10% glycerol). The mixture was incubated at 37℃ overnight. The result was
shown by SDS-PAGE and Coomassie blue staining.
30

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Abstract (if available)

Abstract Proteolysis of the histone H3 N-terminal tail (H3NT) within chromatin is a frequent and conserved epigenetic event in eukaryotes. The proteases responsible for H3NT “clipping” remain largely unknown, which has hampered advances in the field to understand the biological significance of H3NT “clipping”. The goal of my thesis research was to develop an H3NT substrate that could be used in a variety of standard in vitro approaches to greatly accelerate the discovery and investigation of H3NT proteases. Using a bacterial expression system, I generated and purified robust amounts of a recombinant protein that contains 17 tandem repeats of the histone H3 N-terminal tail (rH3NT). My studies validate the utility of the rH3NT substrate in the in vitro H3 cleavage assay and zymography approaches to detect H3NT protease activity using the positive controls, MMP-2 and MMP-9. However, my results also demonstrate that the rH3NT substrate requires refinements to increase the sensitivity of H3NT protease activity detection in the H3 cleavage assay and to improve substrate immobilization for zymography. These forthcoming refinements and considerations are discussed.

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Asset Metadata

Creator Liu, Shulin (author)

Core Title Developing new tools to discover and investigate histone H3 proteases

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Publication Date 07/27/2020

Defense Date 07/08/2020

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag histone H3,histone tail clipping,matrix metalloproteinase,OAI-PMH Harvest,prokaryotic expression,zymography

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Rice, Judd (committee chair), An, Woojin (committee member), Stallcup, Michael (committee member)

Creator Email liushulin@whu.edu.cn,shulinli@usc.edu

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