THE ROLE OF LYSINE-SPECIFICDEMETHYLASE 1 IN GLUCOCORTICOID

RECEPTOR-MEDIATED GENE EXPRESSION

Item Type text; Electronic Thesis

Authors ICENOGLE, ALI LOIS

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 11/03/2021 20:32:23

Link to Item http://hdl.handle.net/10150/613084

ABSTRACT

In order to regulate gene expression, nuclear receptors interact with multiple coregulatory

complexes which contain various enzymatic activities. The glucocorticoid receptor (GR), upon

glucocorticoid (GC) binding, has been shown require the activity of lysine deacetylases

(KDACs) in order to regulate transcription. We have previously demonstrated that KDACs can

function as either transcriptional coactivators or corepressors for GR-target genes, depending on

gene context. Chromatin Immunoprecipitation assays reveal a loss of di-methylated H3K4 at the

transcription start sites of repressed genes when KDACs 1 and 2 are inhibited using Valproic

Acid (VPA). Because no concurrent change in tri-methylated H3K4 was observed, KDACs are

likely to regulate a demethylase to reduce H3K4 di-methylation. Lysine-Specific Demethylase 1

(LSD1) was selected as the candidate, because it is a component of CoREST and NuRD

complexes, which also contain KDACs 1 and 2, and it only demethylates mono- and di-

methylated H3K4. Because LSD1 additionally plays a role in the expression of androgen and

estrogen receptor target genes, the aim of this investigation was to determine the role of Lysine-

Specific Demethylase 1 with respect to GR-mediated gene expression. Through Co-

Immunoprecipitation assays, we confirmed that LSD1 associates with both KDACs 1 and 2. By

monitoring global levels of LSD1 over time, we demonstrated that VPA does not cause changes

in LSD1 expression, nor global changes in mono- and tri-methylated H3K4. Using siRNA-

mediated depletion of LSD1, we determined that LSD1 activity is necessary for transcriptional

repression of GR target genes, but plays little role in activation. However, pharmacological

inhibition of LSD1 using Pargyline, OG-L002, and Bizine did not have any appreciable effects

on the expression of GR activated and repressed genes, though additional assays are needed to

adequately determine the efficacy of the drugs for in vitro studies.

INTRODUCTION

Glucocorticoids (GCs) are steroid hormones whose anti-inflammatory and

immunosuppressive effects have made them effective tools for clinical therapy1,2. The actions of

GCs are mediated through the glucocorticoid receptor (GR), which is a nuclear receptor and

transcription factor. The GR is held in a ligand binding conformation by chaperone proteins in

the cytoplasm of cells. Upon GC binding, the GR dissociates from the chaperones, dimerizes,

and translocates into the nucleus to facilitate gene expression of GR target genes1,2. In order to

control gene expression, nuclear receptors associate with coregulatory complexes. Specifically,

the GR has been shown to interact with enzymes that regulate the acetylation of proteins, lysine

acetyltransferases (KATs) and lysine deacetylases (KDACs)3.

The traditional model of acetylation in transcriptional regulation portrays KATs as

transcriptional coactivators and KDACs as transcriptional corepressors4. However, within the

context of GR, recent studies have demonstrated that KDACs can act as transcriptional

coactivators with respect to GR-activated genes5. Additionally, we have shown that KDAC

activity is necessary for the repression of select GR-target genes, suggesting that KDACs have a

multifaceted role in transcriptional regulation (Item 1).

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A) B)

KDACs act as either transcriptional coactivators or corepressors depending on genetic context.

The mammalian KDAC family is comprised of four classes, based on homology relative to a

prokaryotic counterpart6. The Class I KDACs (KDACs 1, 2, 3, and 8) reside in the nucleus of

cells, where they target histones and other acetylated proteins. Within mammalian cells, KDACs

1 and 2 are present in the Sin3, NuRD, and CoREST complexes, which are thought to have

repressive functions6.

Valproic Acid (VPA) is a Class I-selective KDAC inhibitor that is clinically used to treat

epilepsy and bipolar disorder. Approximately 50% of the patients using this medication

experience metabolic side effects, including weight gain, and impairment to reproductive

function7-9. The significance of this drug with respect to metabolic processes as well as the

known association between KDACs and GR suggests that this drug may negatively impact

normal GR signaling via KDAC inhibition. As a result, this drug is an effective tool for

observing the role of KDAC activity on GR-mediated gene expression5.

Within the nucleosome, histone proteins associate with and dynamically control the

structure of the chromosome. The histone proteins may be subject to post-translational

modifications, thereby changing the area of chromatin accessible to the transcriptional

machinery. Among these histone modifications are acetylation and methylation of lysine

residues. Acetylation of histone H3 as well as methylation of lysine residue 4 of histone H3 is

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Item 1. KDAC Inhibition Impacts GR Mediated Gene Expression at the Transcriptional Level

Hepa1c1c7 cells were treated with 100nM of Dexamethasone (Dex) for 0.5, 1, 2, and 4 hours alone, or

in addition to 5mM of VPA dosed 1 hour prior to Dex treatment (Dex +VPA). RNA isolated from the

transfected cells underwent analysis using RT-qPCR. Intron-exon primers were utilized in order to

measure the expression of nascent transcripts. Error bars signify Standard Error of the Mean (SEM).

Statistical analysis was performed using a paired t test. *, #, p≤0.05; **,##, p≤0.01***, ###,

p≤0.001. Dex samples were compared against the 0 hour control and statistical significance is

represented by *; Dex+VPA samples were compared to their respective time point in the Dex samples,

and statistical significance is represented by #. The results demonstrate that A KDAC inhibition

alleviates GC-mediated transcription or B KDAC inhibition impairs transcriptional activation of GR

target genes.

associated with active transcription17. Additionally, transcriptional regulatory enzymes can alter

acetylation and methylation marks at promoter and enhancer regions of specific genes. Tri-

methylated as well as di-methylated H3K4 is associated with active promoters and enhancers18-

19.

Preliminary chromatin immunoprecipitation (ChIP) data revealed that treatment of mouse

hepatoma cells (Hepa1c1c7) with VPA caused a decrease in di-methylated H3K4 at the

transcription start site of GR repressed genes, without changing the methylation status of tri-

methylated H3K4. The loss of di-methylated H3K4 as a result of KDAC inhibition without a

corresponding increase in tri-methylation suggests that KDACs may interact with a lysine

demethylase. However, ChIP analysis of chromatin treated with VPA demonstrated a significant

increase in di-methylated H3K4 at the response elements (GRE) of GR activated genes, while

causing an increase in tri-methylated H3K4 at specific genes. However, ChIP analysis of

Dexamethasone (GC)-treated cells revealed a slight decrease in H3K4Me2 at the GRE of active

genes suggests GR may recruit a demethylase. Therefore, we wanted to examine which

demethylase is implicated in GR-mediated gene expression, as well as how it functionally

interacts with KDACs in order to accomplish its effects.

In addition to KDACs 1 and 2, Lysine-Specific Demethylase 1 (LSD1) is found in the

NuRD and CoREST complexes10-11. LSD1 exerts repressive function not only through

association with these complexes, but also through demethylation of mono- and di-methylated

H3K4 residues, which serve as marks of active transcription. However, LSD1 activity is

necessary for androgen and estrogen receptor mediated gene expression, indicating that LSD1

may possess a dual function in transcriptional regulation11-13. LSD1’s histone modification

targets, association with complexes containing KDACs 1 and 2, and role in nuclear receptor

signaling suggest that LSD1 may play a role in GR mediated gene expression. We hypothesize

that LSD1 activity is necessary for the expression as well as repression of GR target genes.

In this investigation we confirmed that LSD1 associates with KDACs 1 and 2.

Additionally, we demonstrate that treatment of Hepa1c1c7 cells with VPA does not change the

expression levels of LSD1 nor the global levels of di- and tri-methylated H3K4. LSD1 depletion

restores GC-mediated gene repression in the absence of KDAC activity, suggesting a functional

dynamic between LSD1 and KDACs within this gene context. Conversely, KDAC depletion

minimally impacts expression of GC-repressed genes. Pharmacological inhibition of LSD1 using

Pargyline, OG-L002, and Bizine did not have any effects on GR target gene expression, though

additional assays should be performed to determine the mechanism of impairment within this

system.

MATERIALS AND METHODS

Cell Culture – Murine hepatoma cells (Hepa-1c1c7) were grown in Minimum Essential Medium

α (Gibco, Invitrogen) containing 10% fetal bovine serum (FBS) (Gemini Bio-Products) and

0.1% gentamycin (Gibco, Invitrogen).

RNA Analysis – Cells were seeded in 6-well dishes at 2x105 cells/well and treated the following

day with VPA (5mM), Dex (100nM), or Bizine (10uM). After treatment, the cells were harvested

via lysis using TRIzol (Invitrogen). Total RNA was isolated using the NucleospinRNAII kit

(Clontech). Subsequently, cDNA was synthesized using the iScript cDNA synthesis kit (Bio-

Rad). Real time quantitative polymerase chain reaction (RT-qPCR) was performed using SYBR

Green Master Mix (Bioline) with the Applied Biosystems StepOne instrument. Intron-Exon

primer pairs for each gene tested are illustrated in Table 1. For each gene, the probed Ct values

were normalized against a geometric mean of GAPDH and HPRT1 Ct values to obtain the ΔCt

value for each sample. The primer efficiency for each experiment was calculated using standard

curves, and this was utilized in order to calculate fold change values. The ΔΔCt values were used

to determine the statistical significance using a paired t test (one and two-tailed).

Antibodies and Reagents – In Western Blot Analysis, the antibodies used were anti-H3K4Me2

(Millipore 07-030), anti-H3K4Me3 (Millipore 2591878), anti-LSD1 (Millipore Q2378780),

HDAC1 (Santa Cruz sc-6298), HDAC2 (Santa Cruz sc-81599), and anti-glyceraldehyde

phosphate dehydrogenase (GAPDH) (Santa Cruz sc-25778). Co-Immunoprecipitation antibodies

included anti-α-tubulin (Cell Signaling 29), HDAC1 (Thermo - PA1-860), and

HDAC2 (Bethyl - A300-705A, Thermo - PA1-861). The secondary anti-mouse (115-035-146)

and anti-rabbit (111-035-144) antibodies were purchased from Jackson Immunoresearch and

anti-goat (sc-2056) was purchased from Santa Cruz Biotechnology. Dexamethasone and VPA

were purchased from Sigma-Aldrich. OG-L002 was purchased from Selleck, Pargyline was

purchased from Sigma-Aldrich and Bizine was obtained from Dr. Philip A. Cole (Johns Hopkins

University).

Co-Immunoprecipitation – Hepa1c1c7 cells were seeded in 150mm plates at a density of

2.2X106 cells per plate. After two days cells were harvested, washed with D-PBS and pelleted by

centrifugation at 1500 rpm for 4min at 4°C (Juoan CR3i). The cell pellet was resuspended in D-

PBS and pelleted again. Cell pellet was resuspended in Buffer A [(10mM HEPES pH8.0, 1mM

EDTA pH 8.0, 50mM NaCl, 0.5M sucrose, 10mM sodium phosphate pH 7.3, 5mM DTT, 0.1%

NP-40, phosphatase inhibitors (10nM NaF, 0.1 mM Na3VO4, 25mM β-glycerophosphate), and

protease inhibitor cocktail (Roche)]. For volumes less than 1mL, cells were incubated on ice for

15 minutes. For larger volumes, cells were lysed using a Dounce homogenizer with a loose

fitting pestle. Cell nuclei were pelleted by centrifuging the cell lysate at 1500 rpm for 5min at

4°C (Juoan CR3i). Nuclei were resuspended thoroughly in Buffer B at half the desired volume

[(10mM HEPES pH8.p, 0.1mM EDTA pH 8.0, 100mM NaCl, 25% glycerol, 10mM sodium

phosphate pH 7.3, 5mM DTT, 0.1% NP-40, phosphatase inhibitors (10nM NaF, 0.1 mM

Na3VO4, 25mM β-glycerophosphate), and protease inhibitor cocktail (Roche)]. While vortexing

gently, an equal volume of Buffer B containing 0.6M NaCl was added to obtain a final

concentration of 0.3M NaCl. The extracted nuclei were centrifuged at 30,000xg for 15 minutes.

Supernatant was used immediately or stored at -80°C. Protein A-agarose and protein G-agarose

slurry was used to pre- -buffer [(1M HEPES, 0.5M

EDTA, 1M DTT, 10% Triton-X, with phosphatase inhibitors (10nM NaF, 0.1 mM Na3VO4,

25mM β-glycerophosphate), and protease inhibitor cocktail (Roche)]. To this supernatant 3mg of

either HDAC1, HDAC2, or 5mg anti-GFP antibody and a mixture of protein A-agarose and

protein G-agarose beads (Pierce) was added and rotated overnight at 4oC. The beads were

pelleted and washed three times with IP-buffer. The bound proteins were eluted using 2X SDS-

PAGE Buffer (20% glycerol, 4% sodium dodecyl sulfate, 0.0025% bromo phenol blue, 0.125M

Tris HCl, pH 6.8) followed by incubation at 95oC for 5 minutes prior to separation by SDS-

PAGE.

si-RNA Mediated LSD1 Knockdown – Hepa-1c1c7 cells were plated in 12 or 24 well dishes at a

density of 5x105 cells/well or 2X104 cells/well, respectively, in antibiotic-free MEMα.

DharmaFECT reagent 1 (Dharmacon) was used at a concentration of 0.75μl/well according to

manufacturer’s specifications to transfect cells with siRNA. LSD1 was knocked down using the

corresponding ON-TARGET plus SMARTpool ORF siRNA (Dharmacon) targeting LSD1.

Successful knockdown was confirmed using western blotting. Non-targeting (NT) siRNA was

used as a control.

Western Blotting – Cell lysates were prepared by adding 2X SDS-PAGE Buffer to treated cells.

Cellular proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane

(Biorad) at 400 mA for 2hrs, with the exception of histone modifications, which were transferred

for 4hrs. Membranes were blocked with 2% non-fat dry milk for 1 h, followed by exposure to

primary antibodies at 4oC overnight. After subsequent exposure to appropriate secondary

antibodies, the membrane was washed 3 times with 1X TBS/0.1% Tween-20 solution. The

proteins were visualized using a 1:1 ratio of hydrogen peroxide and luminol (Pierce) with the

Chemi Doc XRS+ molecular imager (Bio-Rad).

PCR Primer Table

Gene (Intron-Exon) Forward Primer Reverse Primer

Hbegf CAGATGCTGGGGTGGGTGAAAT CATTCCTTTCTTTGCTTGGGGTG

Igsf9 CAGAGCCCCTTCCTTCATTCAACA GGCACTCGTACCAACCCTGGTCTT

St5 AGAGCTGAGGATGCACAGATAGCA TATGCCTCTTGGGTAATCGTGGCA

Tns1 TTCTCTCACACGCTTCCGGACTTT

TACAGCACACACAGGCAAGGAACT

Tgm2 TGTCACCAGGGATGAGAGACGG TCCAAATCACACCTCTCCAGGAG

Ampd3 AAGGAGCTTGCAGAGCAGAAGTC CAGCTCCCTCAGGTCTCACAACTAT

Glul GAGCAGAGTGTCTGAACAGCACG ACCCTCCGTGCGCTTACCAG

RESULTS

KDACs 1/2 and LSD1Physically Interact

In our previous studies, we demonstrated that Class I KDAC activity is necessary to

facilitate gene expression of GR target genes and that KDACs 1 and 2 play important role in this

process5. LSD1 has been shown to interact with KDACs 1 and 2 via CoREST and NuRD

complexes10-11. In order to confirm this in our cell line (Hepa1c1c7), we performed co-

immunoprecipitation assays (Fig 1) with antibodies against KDAC1 or KDAC2. Two different

KDAC2 antibodies were utilized in order to verify the efficacy of the antibodies. The results

demonstrate that LSD1 associates with KDACs 1 and 2, as demonstrated by the bands present in

the LSD1 row. The nuclear extract was run as a positive control, to demonstrate the presence of

all three proteins in the sample prior to incorporation of the IP antibodies. The negative control

antibody is α-GFP, which shows no banding except in the KDAC2 blot. Because the band is not

present in the appropriate location corresponding to the kDa of KDAC2, it is likely that this band

is exposure of the heavy chain.

LSD1

HDAC1

HDAC2

IP Antibody

Figure 1. LSD1 associates with KDACs 1 and 2 Hepa1c1c7 cells were plated in 150mm plates at a

density of 2.2X106 cells per plate. After two days, the cells were harvested and underwent Co-

Immunoprecipitation. The final 2X SDS-PAGE buffer supernatant was run on a 10% SDS-PAGE gel and

transferred to a nitrocellulose membrane for 2 hours. The figure reflects the results of the Western Blot

exposure, where the rows reflect the primary antibodies used, and the columns represent the IP

antibodies. Input reflects protein from the original nuclear extract that was untreated with IP antibodies.

Effects of LSD1 Depletion on GR-Mediated Gene Repression

We previously demonstrated that KDAC activity is required for efficient glucocorticoid-

mediated repression of selected target genes. In the preliminary ChIP data, KDAC inhibition

caused a rapid loss of H3K4Me2 at Dex-repressed gene promoters, without a corresponding

increase in H3K4Me3. This suggests a functional interplay between KDACs and LSD1, which

only demethylates di- and mono-methylated H3K4. Because we observed that the KDACi-

induced loss of H3K4Me2 occurred both in the presence and absence of Dex, we hypothesize

that KDAC inhibition activates LSD1, and impairs GC-mediated gene repression. Therefore, we

predicted that depletion of LSD1 will restore Dex-induced repression of GR target genes.

In order to assess the role of LSD1 in GR-mediated repression as well as the functional

interaction between KDACs and LSD1, we used siRNA-mediated knockdown to deplete LSD1,

the synthetic glucocorticoid Dexamethasone (Dex) to induce gene repression, and VPA to inhibit

Class I KDACs (Fig 2). Non-targeting (NT) siRNA was used as the control for this experiment.

To assess gene expression, nascent transcripts were measured using Real-Time quantitative PCR

(RT-qPCR). As expected, in the presence of non-targeting siRNA (NT) Dex repressed

transcription of the Igsf9 and Hbegf promoters and co-treatment with VPA alleviated that

repression. However, when LSD1 is depleted, VPA cotreatment fails to significantly attenuate

Dex-mediated repression. The results suggest that LSD1 activity impairs GC-mediated gene

repression.

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Figure 2. LSD1 depletion recovers Dex-induced gene repression when KDAC activity is

impaired LSD1 depletion was performed using siRNA mediated knockdown, which is reflected in

the LSD1 columns in each graph. Nontargeting (NT) siRNA was utilized as the control for this

experiment. Fourty eight hours following siRNA transfection, cells were treated with either 100nM of

Dexamethasone (Dex) for 1 hour, 5mM of VPA for 2 hours, or a combination of the two (VPA+Dex).

RNA isolated from the transfected cells underwent analysis using RT-qPCR. Intron-exon primers

were utilized in order to measure the expression of nascent transcripts. Error bars signify Standard

Error of the Mean (SEM). Statistical analysis was performed using a paired t test. ***, p≤0.001; ns

denotes no statistical significance.

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Effects of LSD1 Depletion on GR-Mediated Gene activation

In contrast with the repressed genes, ChIP assays of VPA-treated chromatin demonstrated

a significant increase in H3K4Me2 at the Glucocorticoid Response Element (GRE) of GC-

activated genes. VPA treatment had varying effects on H3K4Me3 levels depending on gene

context. This preliminary data indicates that KDACs may interact with a methyltransferase at the

GRE of activated genes. ChIP assays of Dex-treated chromatin showed a slight decrease in

H3K4Me2 observed at the GRE of activated genes, which may suggest that GR recruits or

interacts with LSD1 at these sites. Studies of androgen and estrogen receptors have shown that

LSD1 activity makes a positive contribution to activation of target genes11-13. As a result, we

asked whether LSD1 activity is necessary for GC-induced gene activation. We also wanted to

determine whether it contributes to impaired GR transactivation upon KDAC inhibition.

In order to evaluate the role of LSD1 in GR-mediated gene activation, we depleted LSD1

using siRNA, used Dex to induce gene activation in our system, and treated with VPA to inhibit

Class I KDACs (Fig 3). The corresponding changes in gene expression for each condition were

measured via RT-qPCR analysis of the nascent transcripts. The results of the investigation

demonstrate two distinct results. For the majority of activated genes, depletion of LSD1 did not

cause an impairment of Dex-induced activation relative to non-targeting siRNA (NT) (Fig

3a,b,c,d). However, LSD1 depletion demonstrated a slight impairment of Glul transactivation

relative to NT (Fig 3e). The results suggest that LSD1 activity is not required for GC-mediated

activation of genes that are sensitive to KDAC inhibition.

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Effects of KDAC Inhibition on Global Levels of Histone Modifications

The ChIP assays evaluating the impact of VPA on histone modifications analyzed local

levels of di- and tri-methylated H3K4 at particular genes. In order to evaluate whether VPA

induces a change in global levels of di- and tri-methylated H3K4 as well, cells were treated with

VPA, and the overall H3K4 methylation status was measured via Western Blot analysis (Figure

4). In order to verify that any observed changes in H3K4 methylation are not due to a change in

levels of histone H3, the total Histone H3 was monitored over the time course as well. The

results of the VPA time course indicate that global levels of H3K4Me2 do not significantly

increase over the course of the five hours of treatment (Fig 4a,b). Similarly, global levels of

H3K4Me3 did not change significantly over the course of treatment (Fig 4c,d). The data indicate

that, VPA-induced changes in H3K4 methylation occur in local regions of chromatin rather that

globally.

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Figure 3. LSD1 depletion does not impair Dex-induced gene activation LSD1 depletion was

performed using siRNA mediated knockdown, which is reflected in the LSD1 columns in each graph.

Nontargeting (NT) siRNA was utilized as the control for this experiment. Fourty eight hours

following siRNA transfection, cells were treated with either 100nM of Dex for 1 hour, 5mM of VPA

for 2 hours, or a combination of the two (VPA+Dex). RNA isolated from the transfected cells

underwent analysis using RT-qPCR. Intron-exon primers were utilized in order to measure the

expression of nascent transcripts. Error bars signify Standard Error of the Mean (SEM). Statistical

analysis was performed using a paired t test. *, p≤0.05. Two different responses were observed:

graphs A-D demonstrated no significant change between the expressions of the Dex samples between

NT and LSD1 depleted conditions, graph E reflected a decrease in expression with the LSD1 depleted

sample relative to NT.

*

VPA, h 0 2 5

VPA, h 0 2 5

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Figure 4. KDAC inhibition does not change global levels of histone modifications Hepa1c1c7 cells

were seeded at 2.2X106 cells per well in 6 well plates. The following day, cells were treated with

5mM of VPA for either 2 or 5 hours. The 0 hour sample reflects cells untreated with VPA. Cell

lysates were washed with PBS and harvested using 2X SDS-Buffer. Cell lysates were run on a 10%

SDS-PAGE gel and transferred onto a nitrocellulose membrane for four hours. The exposed Western

blots are demonstrated by images A and C. Volume analysis was performed using Image Lab

software, and the H3K4Me2 and H3K4Me3 protein volumes were normalized relative to Histone H3.

The 2 and 5 hour treatments were then normalized to the 0 hour time point. The histone modification

quantitations are reflected in graphs B and D.

Effects of KDAC Inhibition on LSD1 Expression

The preliminary ChIP data demonstrated a significant decrease in di-methylated H3K4 at

the promoters of GR repressed genes upon introduction of VPA. Though the results of the LSD1

knockdown demonstrate that LSD1 depletion restores GC-mediated repression in the absence of

KDAC activity, it is unclear as to how KDACs may impact LSD1 function. In order to determine

whether the observed decrease in H3K4Me2 is the result of LSD1 activation or the result of

upregulation of LSD1 expression, we analyzed the global changes in LSD1 expression with VPA

treatment (Fig 5). The results of the analysis indicate that VPA does not affect LSD1 expression

in the cells.

VPA, h 0 2 5

Pharmacological Inhibition of LSD1 has Negligible Effects on Gene Expression

The decrease in H3K4Me2 at the promoters of Dex-repressed genes in the absence of

KDAC activity suggests that KDACs functionally interact with LSD1. Additionally, the LSD1

knockdown demonstrated that depletion of LSD1 restores gene repression in the absence of

KDAC activity. This suggests that LSD1 activation is incompatible with Dex-induced repression

at the genes tested. On the other hand, Dex caused a slight decrease in H3K4Me2, yet

knockdown of LSD1 did not attenuate Dex-induced gene activation. Nonetheless, it is possible

that siRNA-mediated knockdown of LSD1 impacts the stability of the entire CoREST complex,

in which LSD1, and KDACs 1 and 2 are core components14-15. As an alternative route to gauge

the impact of LSD1 on GR-mediated gene expression, pharmacological inhibition of LSD1 was

employed (Fig 6). We used three different LSD1 inhibitors – Bizine (Fig 6d), OG-L002 (Fig 6e),

and Pargyline (Fig 6f) – at concentrations shown by others to impair LSD1 activity. Cells were

LSD1

GADPH

0h

r2h

r5h

r

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A)

B)

Figure 5. KDAC inhibition does not change expression of LSD1 Hepa1c1c7 cells were seeded at

2.2X106 cells per well in 6 well plates. The following day, cells were treated with 5mM of VPA for

either 2 or 5 hours. The 0 hour sample reflects cells untreated with VPA. Cell lysates were washed

with PBS and harvested using 2X SDS-Buffer. Cell lysates were run on a 10% SDS-PAGE gel and

transferred onto a nitrocellulose membrane for four hours. The exposed Western blots are

demonstrated by image A. Volume analysis was performed using Image Lab software, and the LSD1

protein volumes were normalized relative to GAPDH. The 2 and 5 hour treatments were then

normalized to the 0 hour time point. The histone modification quantitations are reflected in graph B.

treated with or without the LSD1 inhibitors in the presence or absence of Dex. Expression of

GR activated and repressed genes, represented by Ampd3 and Hbegf, respectively, was

examined. In terms of the representative activated genes, LSD1 inhibition did not impair Dex-

induced gene activation. Likewise, LSD1 inhibition in the absence of KDAC activity did not

recover GC-induced gene repression.

Co

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Dex+V

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+D

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T re a tm e n t:

A)

B)

C)

D)

E)

Effects of Pharmacological Inhibition of LSD1 on Global Levels of LSD1 and Histone

Modifications

As demonstrated in Figure 6, the treatment of Hepa1c1c7 cells with Bizine did not impact

the gene expression of GR target genes. In order to determine whether Bizine was effectively

targeting LSD1, it was necessary to develop an assay to assess the global levels of di-methylated

and tri-methylated H3K4. To attempt to understand whether Bizine influences global histone

modifications, we examined cell extracts treated with 10 μM of Bizine for 2 and 6 hours (Figure

7). Treatment times were constructed based on the previous literature which states that Bizine

effectively causes an increase in di-methylated H3K4 by 6 hours16. Additionally, LSD1 levels

were monitored over the course of Bizine treatment in order to verify that the drug was not

impacting LSD1 expression, or causing protein degradation. The results demonstrate that LSD1

expression levels do not change throughout the course of Bizine treatment (Fig 7a,b).

Additionally, levels of histone modifications do not significantly change throughout the course

of treatment (Fig 7c,d,e,f).

Co

ntr

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ylin

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ylin

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0 .0

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T re a tm e n t:

Figure 6. Pharmacological inhibition of LSD1 does not impact GR-mediated gene expression

The structures of the LSD1 inhibitors are reflected in images A-C. A is the structure of OG-L002, B is

the structure of Pargyline, and C is the structure of Bizine. Hepa1c1c7 cells were seeded at 2.2X106

cells per well in 6 well plates. The following day, cells were treated with either 100nM of Dex for 1

hours, 5mM of VPA for 2 hours, the respective LSD1 inhibitor, or a combination. In terms of LSD1

inhibitor treatment, cells were dosed with either D) 10 μM of Bizine for 2 hours, E) 50 uM (Ampd3)

or 100 uM (Hbegf) of OG-L002 for 24 hours, or F) 3mM of Pargyline for 24 hours. Cells were then

harvested, and isolated RNA underwent RT-qPCR using intron-exon primers to measure expression of

nascent transcripts. For each of the LSD1 inhibitors used, a representative GR activated (Ampd3) and

repressed (Hbegf) gene was analyzed. Error bars represent SEM. Statistical analysis using a paired t

test was performed, but none of the results demonstrated significance.

F)

Bizine, h 0 2 6

Bizine, h 0 2 6

Bizine, h 0 2 6

LSD1

GADPH

H3K4Me2

Histone H3

H3K4Me3

Histone H3

0 2 6

0 .0

0 .5

1 .0

1 .5

H 3 K 4 M e 2

B iz in e T re a tm e n t

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0 2 6

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1 .5

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B iz in e T re a tm e n t

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0 2 6

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0 .5

1 .0

1 .5

L S D 1

B iz in e T re a tm e n t

Fo

ld C

ha

ng

e

A)

C)

B)

D)

E)

F)

Figure 7. Pharmacological inhibition of LSD1 does not impact global levels of LSD1 or histone

modifications Hepa 1c1c7 cells were seeded at 2.2X106 cells per well in 6 well plates. The following

day, cells were dosed with 10 μM of Bizine for 2 hours and 6 hours. The 0 hour treatment reflects

cells untreated with Bizine. Cells were then harvested with 2X SDS-PAGE Buffer and subjected to

Western Blot Analysis. A) LSD1 levels were monitored over the course of Bizine treatment. B)

Volume analysis performed using Image Lab Software was performed. Each biological replicate was

averaged in order to yield the results depicted in the graph. The LSD1 protein bands were normalized

relative to GAPDH, and the 2 and 6 hour time points were then normalized relative to the 0 hour. C)

Di-methylated H3K4 levels were monitored over the course of Bizine treatment. D) Volume analysis

performed using Image Lab Software was performed. Each biological replicate was averaged in order

to yield the results depicted in the graph. The H3K4Me2 protein bands were normalized relative to

Histone H3, and the 2 and 6 hour time points were then normalized relative to the 0 hour. E) Tri-

methylated H3K4 levels were monitored over the course of Bizine treatment. F) Volume analysis

performed using Image Lab Software was performed. Each biological replicate was averaged in order

to yield the results depicted in the graph. The H3K4Me3 protein bands were normalized relative to

Histone H3, and the 2 and 6 hour time points were then normalized relative to the 0 hour.

DISCUSSION

In order to regulate gene expression, nuclear receptors often require coregulatory

enzymes. Though a significant body of literature is centered on elucidating the function of these

coenzymes with respect to nuclear receptor signaling, many of the components and mechanisms

of coregulatory complexes are still unknown. In the context of the glucocorticoid receptor, we

have previously demonstrated that KDAC activity is necessary for GR-mediated gene

activation5. By examining the H3K4 methylation status of various GR target genes in the

absence of KDAC activity, we concluded that KDACs functionally interact with lysine

deacetylases and lysine methyltransferases. When cells were treated with VPA, we observed a

loss of di-methylated H3K4 at the promoters of GR repressed genes with no corresponding

change in tri-methylated H3K4, which suggests KDACs may interact with a lysine demethylase

that preferentially targets H3K4Me2. Additionally, Dex-treated cells demonstrated a slight loss

of di-methylated H3K4 at the Glucocorticoid Response Elements of GR-activated genes,

demonstrating that GR may recruit the demethylase to facilitate gene expression.

In CoREST as well as NuRD complexes, KDACs 1 and 2 have been shown to associate

with Lysine-Specific Demethylase 1 (LSD1)10-11. Additionally, LSD1 possesses a regulatory role

in androgen and estrogen receptor-mediated gene expression11-13. Due to the body of literature

which portray LSD1 as a coactivator of steroid receptor signaling as well as an integral

component of complexes containing KDACs 1 and 2, we hypothesized that LSD1 has a

functional role in GR-mediated gene expression.

Co-Immunoprecipitation assays (Fig. 1) confirmed that KDACs 1 and 2 associate with

LSD1 in our cellular system. However, the Co-IP alone does not illustrate the other proteins

associated with the complex. In future experiments, a Co-IP examining whether LSD1 is

associating with CoREST or NURD complex subunits may further our knowledge of the

regulatory enzymes associated with LSD1.

If LSD1 activity is required for GR-mediated gene expression, then the absence of LSD1

will influence gene expression levels. Therefore, we utilized siRNA-mediated knockdown of

LSD1 to simulate transcription in the absence of LSD1. When LSD1 is expressed and KDACs

are active, Dex induces repression of select GR gene targets (Fig 2). When KDAC activity is

impaired by KDACi, this repression is significantly alleviated. However, upon LSD1 depletion,

gene repression by Dex is recovered in the presence of KDACi. This suggests that LSD1

activation acts against Dex-mediated gene repression. When KDACs are functional, they are

likely suppressing LSD1 activity and keeping it from impairing transcription, as demonstrated by

Dex repression of genes when KDACs are active. The results establish evidence for the

functional relationship between LSD1 and KDACs with regards to GR-mediated gene

repression.

While the preliminary ChIP data revealed that KDACi caused a loss of di-methylated

H3K4 at the promoters of repressed genes, VPA caused a significant increase in di-methylated

H3K4 at the GREs of activated genes. This suggests that KDACs may regulate a

methyltransferase as opposed to a demethylase in the context of active genes. However, Dex

treatment resulted in a slight loss of di-methylated H3K4, indicating that GR may activate or

recruit a demethylase to facilitate gene activation. Within the context of active genes, we

predicted that LSD1 activity is required for GR-mediated gene activation, but LSD1 may not

interact KDACs in order to do so. To extend this analysis to GR-activated genes, we analyzed

the LSD1 knockdown for the expression of this group of genes. The results demonstrated two

different gene responses. For the majority of genes, activation was not significantly impaired by

LSD1 depletion (Fig 3a,b,c,d). Yet, LSD1 depletion slightly attenuated the activation of one

gene, Glul (Fig 3e). This finding indicates that LSD1 has minimal, if any, functional role in the

activation of the GR target genes tested in this investigation. The role LSD1 plays in GR-

mediated transcription may depend on genetic context, as shown in the contrasting results

obtained between activated and repressed genes.

The initial ChIP analysis of H3K4 methylation largely focused on the promoters of

specific GR target genes. To determine whether the change in histone methylation status is

primarily localized, we examined the global levels of H3K4Me2 and H3K4Me3 when VPA is

administered (Fig 4). For both modifications, there was no significant change relative to control

over the course of VPA treatment. The results indicate that KDAC inhibition causes changes in

the methylation status of H3K4 locally at the promoters of individual genes as opposed to

genome-wide. In order to ensure KDAC inhibition resulted in the activation of LSD1 as opposed

to upregulation, we examined whether VPA treatment impacted LSD1 expression levels (Fig 5).

Over the course of VPA treatment, there was no observable change in the global levels of LSD1,

indicating that VPA does not cause cause decreased H3K4Me2 through LSD1 upregulation.

The structural integrity of transcriptional regulatory complexes depends on the presence

of integral subunits. With respect to the LSD1-CoREST complex, knockdown of either LSD1 or

Co-REST results in degradation of the other protein15-16. This suggests that depletion of one of

the core components of this complex compromises the structural integrity, and the remaining

coenzymes are subsequently degraded. Taking this notion into account, LSD1 depletion may

decrease cellular levels of the intact CoREST complex resulting in a decrease in complex

association with the Dex-repressed gene promoters. In order to assess the role of LSD1 in GR-

mediated gene expression without compromising the structural integrity of the complexes, we

used pharmacological inhibitors to impair LSD1 activity (Fig 6). The inhibitors used were

Bizine, OG-L002, and Pargyline (Fig 6a,b,c). The results demonstrated that none of inhibitors

significantly impacted gene expression. Taking into consideration the results of the siRNA

mediated knockdown, we expected that gene activation, represented by Ampd3, would not be

impaired in the absence of LSD1 activity. However, the knockdown data predicted that

inhibition of LSD1 would result in the recovery of Dex-induced repression in the absence of

KDAC activity. Nevertheless, the data reflect the maintenance of alleviation of repression. The

deviation between the knockdown and inhibitor data suggest that the inhibitors are not

successfully impairing LSD1 or that LSD1 depletion is causing a loss of complex structural

integrity that is not achieved with pharmacological inhibitors but that contributes to Dex-

mediated repression.

In order to assess the efficacy of the pharmacological inhibitors, we conducted an assay

to measure global histone H3 methylation levels over the course of Bizine treatment (Fig 7). The

results indicate that they do not change with Bizine treatment (Fig 7a,b,c,d). Additionally, the

LSD1 expression levels were also monitored over the course of treatment in order to assess

whether Bizine was impairing activity or expression of LSD1. The data indicate that Bizine does

not affect LSD1 expression levels (Fig 7e,f). However, global levels of histone modifications

may not be a sufficient assay for analyzing the efficacy of the pharmacological inhibitors. To

move forward with these inhibitors, it may be necessary to perform a ChIP assay, in which

H3KMe2 and H3K4Me3 levels are measured at the promoters of GR target genes when cells are

treated with Bizine in the presence and absence of KDACi. If bizine effectively inhibits LSD1,

it should block the KDACi-induced decrease in H3K4Me2.

VPA is clinically utilized in order to treat epilepsy and bipolar disorder7. As VPA and

other drug targets of coregulatory enzymes become widely available, it is necessary to determine

their physiological effects. VPA impacts GR signaling in ways that significantly change the

expression of GR target genes. By understanding the composition of regulatory complexes, it is

possible to design more effective therapeutics and predict the side effects.

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