Estrogen receptor a mediated long rang chromatin interactions at the ret gene locus in breast cancer

ESTROGEN RECEPTOR α MEDIATED LONG
RANGE CHROMATIN INTERACTIONS AT THE
RET GENE LOCUS IN BREAST CANCER

LIN ZHENHUA

NATIONAL UNIVERSITY OF SINGAPORE
10
20
2010


Acknowledgement
I would like to express my gratitude to all lab members in Cancer Biology and
Pharmacology Lab 3 at the Genome Institute of Singapore. My sincere appreciation
goes to Dr Edwin Cheung for his patience and guidance throughout the project. In
addition I would like to thank Dr Ng Huck Hui for introducing me into DBS and his
guidance during my course of study. My special thanks go to Dr Liu Mei Hui for her
advice on the 3C assay and Ms Tan Si Kee for her assistance with the co-factor
studies. Many thanks go to other members in my lab who has helped me in one way
or another. Without the group in CB3, I will not be able to finish my project and thesis
so smoothly.


Table of Contents
SUMMARY

i

LIST OF TABLES

iii

LIST OF FIGURES

iv

LIST OF ABBREVIATIONS

vi

Chapter 1 Background

1

1.1 Breast cancer and estrogen

1

1.1.1 Breast cancer and estrogen

1

1.1.2 Estrogen and its role in human physiology

2

1.1.3 Estrogen and the estrogen receptor

2

1.1.4 Molecular mechanism of the estrogen receptor

3

1.2 RET gene

4

1.2.1 RET and its isoforms

4

1.2.2 RET gene and its role in human physiology

6

1.3 Long range chromatin interactions

7

1.3.1 Estrogen receptor binding sites in breast cancer

7

1.3.2 Methods to study long range chromatin interactions

8

1.3.3

Long range chromatin interactions of the estrogen receptor

11

1.4 Aims and objectives of the study

12

Chapter 2 Material and Methods

13


2.1 Plasmids construction

13

2.1.1 PCR amplification

13

2.1.2 Homologous recombination

15

2.2 Mutagenesis

15

2.3 Cell culture, transfection and luciferase assays

18

2.3.1 Cell culture

18

2.3.2 Transient transfection and luciferase/renilla dual reporter assay

18

precipitation assay
2.4 Chromatin immuno
immunoprecipitation

19

2.5 Chromosome conformation capture

22

2.5.1 3C assay

22

2.5.2 Primer design and qPCR

23

2.5.3 BAC control

25

2.6 RNA expression

26

2.
2.77 Protein expression

28

2.7.1 Total protein extraction

28

2.7.2 SDS-polyacrylamide gel electrophoresis

28

2.7.3 Western Blotting

29

2.8 siRNA knockdown

29

2.9 Evolutionary conservation analysis

30

Chapter 3 Results

31

3.1 The RET proto-oncogene is up-regulated by estrogen

31


3.1.1 RET mRNA and protein expression level is upregulated after E2 treatment

31

3.1.2 Effect of ERα knockdown on RET mRNA expression

35

3.1.3 The RET gene is a primary target of ERα

37

3.2 RET gene is regulated by E2 through two imperfect EREs

39

3.2.1 ERα binds to six estrogen receptor biding sites at the RET gene locus

39

3.2.2 Two of the six estrogen receptor binding sites are functional

42

3.2.3 Mutagenesis confirmed the function of EREs in these two estrogen
receptor binding sites

44

α binding sites interact with the transcription start
3.3 Two ER
ERα
site

46

3.3.1 3C assay detected three E2 dependent long range chromatin interactions
around the RET
3.3.2 Long range chromatin interactions at the RET gene requires ERα

46
50

3.4 Role of other co-factors in long range chromatin interactions
around RET

52

3.4.1 Binding of other co-factors at the RET locus through ChIP

52

3.4.2 Mutagenesis of the co-factor motif decreased ERα enhancer ability

56

3.4.3 AP2γ is required for RET gene expression

58

3.4.4 Recruitment of ERα to the estrogen receptor binding sites of RET is 60
dependent on AP2γ
3.4.5 AP2γ affects ERα mediated long range chromatin interactions

3.5 Conservation of the RET gene
gene’’s ERα binding sites

62
64


Chapter 4 Discussion

66

α mediated long range
4.1 RET is E2 regulated through ER
ERα

chromatin interactions

66

4.2 RET gene plays a functional role in human breast cancers

69

2γ functions as a pioneer factor for ER
α response pathway
4.3 AP
AP2
ERα

70

Chapter 5 Conclusion

72

References

73


SUMMARY
Estrogens function as the primary female sex hormones in women of reproductive age.
It promotes the development of female secondary sexual characteristics, and is an
essential part of a woman’s reproductive process. Recent studies indicate that about
80% of breast cancers, once established, are estrogen dependent. These are known as

hormone-sensitive or hormone-receptor-positive cancers. ERα is the key transcription
regulator of this breast cancer progression. It is over-expressed in around 70% of
breast cancer cases and estrogen has been shown to stimulate proliferation of
mammary cells.

Using high-throughput ChIP (Chromatin ImmunoPrecipitation)-based technology,
such as ChIP-Seq (ChIP process followed by sequencing), we generated a global map
of ERα binding sites in the genome of MCF7 breast cancer cell line. From this dataset,
we identified two ERα binding sites near the RET (rearrangement during transfection)
gene. RET is a proto-oncogene that encodes a receptor tyrosine kinase. It has been
shown to be involved in human papillary thyroid tumors and in multiple endocrine
neoplasia type 2. Previous studies have shown the RET gene is expressed in primary
breast tumors and cell lines. In MCF7 cells, the expression of RET, the activation of
its downstream signaling pathways, and the increase of anchor-independent
proliferation have been shown to be estrogen dependent. In addition, RET expression
is up-regulated by estrogen treatment and knock down of ERα down-regulates RET.

i


We verified that two ERα binding sites within the RET locus, which are located 50kb
upstream and 35kb downstream of the transcription start site, are recruited upon
estrogen stimuli. Using Chromosome Conformation Capture assay, we showed that
these two ERα binding sites are brought in close proximity with each other and with
the promoter region of the RET gene in an estrogen dependent manner. Knock down
of ERα disrupted this long-range interaction. In addition, we showed that
co-regulatory factors, such as FoxA1, cJun, and AP2γ are recruited to these two ERα
binding sites. Among them, AP2γ knock-down resulted in a decrease of RET
expression and a concomitant decrease in long range chromatin interaction. Taken
together, these results suggest that ERα collaborates with other DNA binding

transcription factors to form chromatin loops which directly regulate the transcription
of the RET gene in breast cancer cells.

ii


LIST OF TABLES
Table 1: Primer sets used to amplify the 6 ERBSs.

14

Table 2: Primers used to introduce mutations into transcription factor binding motif.

17

Table 3: Primer sets used to detect the 6 ERBSs.

21

Table 4: Primer sets used for the 3C assay.

24

Table 5: Primer sets used to detect mRNA expression level.

27

Table 6: Revolutionary conservation analysis of three ERE sites around RET gene.

65

iii


LIST OF FIGURES
Fig 1: RET mRNA expression levels with E2 treatment in MCF7 cells.

32

Fig 2: RET51 protein expression level with E2 treatment in MCF7 cells.

34

Fig 3: ERα, RET9 and RET51 mRNA expression level after ERα siRNA

36

knockdown.
Fig 4: RET9 and RET51 mRNA expression level with cycloheximide treatment.

38

Fig 5: ERBSs of the RET gene locus.

40

Fig 6: Recruitment of ERα at six ERBSs of the RET gene locus.

41

Fig 7: Functional analysis of the 6 ERBS through transient transfection.

43

Fig 8: Mutation analysis of ERBS 1 and ERBS 6 through transfection.

45

Fig 9: Overview of ERBS location and primers designed for 3C assay.

48

Fig 10: Long range chromatin interaction at the RET gene locus through 3C.

49

Fig 11: Long range chromatin interaction at the RET gene locus after ERα siRNA

51

knockdown.
Fig 12: Prediction of other co-factor at ERBS 1 and ERBS 6 through motif

53

analysis.
Fig 13: cJun and FoxA1 binding at the RET locus.

54

Fig 14: AP2γ binding at the RET locus.

55

Fig 15: Mutation analysis of ERBS with co-factor motif mutations.

57

Fig 16: Protein and mRNA expression of RET after AP2γ siRNA knockdown.

59

Fig 17: ERα binding at the RET locus after AP2γ knockdown.

61

Fig 18: Long range chromatin interaction at the RET gene locus after AP2γ
siRNA knockdown.

63
iv


Fig 19: Overview of ChIA-PET interaction around the RET gene locus.

68

v

LIST OF ABBREVIATIONS
3C: Chromosome Conformation Capture
3D: Deconvolution of DNA interaction by DNA selection and ligation
BAC: Bacterial Artificial Chromosome
ChIA-PET: Chromatin Interaction Analysis by Paired-End Tag
ChIP: Chromatin Immunoprecipitation
ChIP-Seq: ChIP process followed by sequencing
CHX: Cycloheximide
E2: Estradiol
ER: Estrogen Receptor
ERBS: Estrogen Receptor Binding Site
ERE: Estrogen Response Element
GDNF: Glial cell line-Derived Neurotrophic Factor
PCR: Polymerase Chain Reaction
RET: Rearrangement during Transfection
RLU: Relative Luciferase Unit
TSS: Transcription Start Site

vi


Chapter 1 Background
1.1 Breast Cancer and Estrogen

1.1.1

Breast cancer and estrogen

Breast cancer is the second ranking cancer worldwide and it is the fifth most common
cause of cancer death (Breast Cancer Facts & Figures 2009-2010, American Cancer

Society, Atlanta, Georgia). With decades of molecular pathology research and clinical
trials, breast cancer is also one of the most well studied cancer types now and its
survival rate after therapy is increasing (Wooster and Weber 2003). Established breast
cancer cell lines, such as MCF7, is now a common model for the study of breast
cancer.

Based on the dependency of hormones, breast cancers can be generally divided into
two groups, hormone-sensitive and hormone-insensitive breast cancers. The first
group, which constitutes about 80% of all breast cancers, is also known as
hormone-receptor-positive breast cancers. Such cancers, once established, rely on the
hormone estrogen to grow (Perou, Sorlie et al. 2000; Sorlie, Perou et al. 2001; van de
Vijver, He et al. 2002; Yager and Davidson 2006; Sadler, Pugazhendhi et al. 2009).

1


1.1.2

Estrogen and its role in human physiology

Estrogens (or oestrogens) are a group of steroid compounds, named for their
importance in the estrous cycle (Nelson and Bulun 2001; DeNardo, Kim et al. 2005).
Estrogen functions as the primary female sex hormone. There are three major
naturally occurring estrogens in women: estrone (E1), estradiol (E2), and estriol (E3)
(Dahlman-Wright, Cavailles et al. 2006). Even though estrogens are present in both
men and women, they are usually present at significantly higher levels in women of
reproductive age. They promote the development of female secondary sexual
characteristics, such as breasts, and are also involved in the thickening of the
endometrium and in the regulation of the menstrual cycle (Yager and Davidson 2006).
In males, estrogen regulates specific functions of the reproductive system that are

important in the maturation of sperm and may be necessary for a healthy libido (Hess,
Bunick et al. 1997).

1.1.3

Estrogen and the estrogen receptor

Estrogen functions through binding to the estrogen receptors. The estrogen receptor
(ER) belongs to a subfamily of the nuclear receptor superfamily (Nilsson, Makela et
al. 2001). There are two isoforms of the ER, ERα and ERβ, and each is encoded by a
separate gene, ESR1 and ESR2, respectively (Leung, Mak et al. 2006). Despite this,
ERα and ERβ show significant overall sequence homology (Ascenzi, Bocedi et al.
2


2006).

Like other members of the nuclear receptor superfamily, the ERs have three major
domains, the Activation Function domain, the DNA Binding Domain and the Ligand
Binding Domain (Shiau, Barstad et al. 1998). After estrogen activation, the estrogen
receptors may form 3 different dimers, ERα or ERβ homodimers or ERαβ
heterodimers (Couse, Lindzey et al. 1997; Li, Huang et al. 2004). Across various cell
types, the ERα homodimer is the most common one and is over-expressed in 70% of
breast cancer cases (Deroo and Korach 2006). Estrogen activates ERα and stimulate
the proliferation of mammary cells (Fabian and Kimler 2005). Indeed, ERα has been
shown to regulate important cell cycle genes, such as Cyclin D1 and DNA
methylation genes, such as O-6-methylguanine-DNA methyltransferase (Metivier,
Penot et al. 2003; Bjornstrom and Sjoberg 2005; Levin 2005).

1.1.4 Molecular mechanism of the estrogen receptor

In the classical model, ERs are activated through ligand binding. Binding of estrogen
to the receptors leads to homodimerization. The homodimers subsequently bind to
specific response elements known as estrogen response elements (EREs) located in
the promoters of the target genes to assist transcription (Nilsson, Makela et al. 2001).
Estrogen binding also induces conformational changes within the ligand binding
domain of the ERs, and this change allows coactivator proteins to be recruited
3


(Rosenfeld and Glass 2001). In total, one third of the genes in humans that are
regulated by ERs can be activated in this ERE-dependent manner (O'Lone, Frith et al.
2004).

Besides this classical model, ERs can regulate gene expression without binding
directly to DNA by modulating the function of other transcription factors through
protein-protein interactions (Gottlicher, Heck et al. 1998). Several genes are activated
by E2 through the interaction of ERs with cJun and cFos proteins at AP-1 binding
sites within the promoter of genes such as IGF-I (Umayahara, Kawamori et al. 1994)
and cyclin D1 (Sabbah, Courilleau et al. 1999; Liu, Albanese et al. 2002). Besides
AP-1 binding sites, ERs also regulate GC-rich promoter regions with the Sp1
transcription factor (Porter, Saville et al. 1997; Li, Briggs et al. 2001). These
ERE-independent actions mainly rely on the tethering of ERs to other DNA binding
transcription factors so as to enhance ER transcriptional regulation (Bjornstrom and
Sjoberg 2005).

1.2 RET Gene

1.2.1

RET and its isoforms

RET proto-oncogene was named because of its Rearrangement during Transfection
(Takahashi, Ritz et al. 1985).

The DNA sequence of this gene was originally found
4


to be rearranged within 3T3 fibroblast cell line following its transfection with DNA
taken from human lymphoma cells. In human, RET gene is located in chromosome 10
(10q11.2) and contains 21 exons (Takahashi 1988; Takahashi, Buma et al. 1988;
Ishizaka, Itoh et al. 1989).

The RET gene encodes a receptor tyrosine kinase (RTK) which belongs to the glial
cell line-derived neurotrophic factor (GDNF) family of extracellular signaling
molecules (Durbec, Marcos-Gutierrez et al. 1996; Trupp, Arenas et al. 1996; Baloh,
Tansey et al. 1998). Alternative splicing results in 3 different isoforms of RET, RET9,
RET43 and RET51, based on the 9, 43 and 51 amino acids in their C-terminal tail
respectively (Tahira, Ishizaka et al. 1990; Myers, Eng et al. 1995; de Graaff, Srinivas
et al. 2001). RET43 is seldom found in human (Myers, Eng et al. 1995).

The RET protein is divided into 3 domains. In the N-terminal extracellular domain
there are four cadherin-like repeats and a cysteine-rich region. The hydrophobic
transmembrane domain and the cytoplasmic tyrosine kinase domain are separated by
an insertion of 27 amino acids. Within their cytoplasmic domains, there are 16
tyrosines (Tyr) in RET9 and 18 tyrosines in RET51 (Hayashi, Iwashita et al. 2001;
Kurokawa, Iwashita et al. 2001). Tyr1090 and Tyr1096 are unique for RET51 (Knauf,
Kuroda et al. 2003; Kawamoto, Takeda et al. 2004).

5


1.2.2 RET gene and its role in human physiology

Mice deficient in GDNF, GFRα1 or the RET protein exhibit severe defects in kidney
and enteric nervous system development (Trupp, Scott et al. 1999; Lee, Chan et al.
2002). This implicates that RET signal transduction is a key pathway in the
development of normal kidneys and the enteric nervous system. RET loss of function
mutations are associated with the development of Hirschsprung's disease, while gain
of function mutations are associated with the development of various types of human
cancer, including medullar thyroid carcinoma, multiple endocrine neoplasias type 2A
and 2B, phaeochromocytoma and parathyroid tumors (Ishizaka, Itoh et al. 1989;
Donis-Keller, Dou et al. 1993; Mulligan, Kwok et al. 1993; Edery, Lyonnet et al. 1994;
Hofstra, Landsvater et al. 1994; Romeo, Ronchetto et al. 1994; Eng 1999).

Recently researchers have also demonstrated the role of RET in tumors progression
from non-neuroendocrine origin. Furthermore, detection of RET mutations in
pancreatic cancer and the over expression of genes in the RET RTK pathway in breast
tumor cell lines suggest that RET have important roles in the regulation of cancer
growth and progression (Hayashi, Ichihara et al. 2000; Dechant 2002; Tsui-Pierchala,
Milbrandt et al. 2002; Sawai, Okada et al. 2005; Zeng, Cheng et al. 2008).

6


1.3 Long Range Chromatin Interactions

1.3.1 Estrogen receptor binding sites in breast cancer

The human genome is comprised of 23 pairs of chromosome with a total of 3 billion
base pairs (Lander, Linton et al. 2001; Venter, Adams et al. 2001). However, only
1.5% of the genome encodes for about 23,000 of all protein-coding genes. Within the
non-coding sequences, there are many different kinds of regulatory elements which
provide crucial control of gene expression. These elements include insulators,
boundary elements and transcription factor binding sites (Maston, Evans et al. 2006).

One of the important functions of these regulatory elements is their role as
recruitment sites for protein factor to carry out their regulatory functions (West and
Fraser 2005). Chromatin Immunoprecipitation (ChIP) is widely used to detect such
protein-DNA interactions and ChIP-Seq provides us with the tools to map the position
of these regulatory sites across the genome (Kuo and Allis 1999). For example,
traditional ChIP assay using a specific antibody against ER isolates chromatin
fragments bound by the receptor. By performing traditional or quantitative PCR with
specific primers, estrogen receptors binding at specific locations in the genome can be
easily detected. By coupling ChIP with massive sequencing technology, the whole
pool of estrogen binding fragments can be sequenced and mapped back to the human
genome revealing the exact positions of estrogen receptor binding site (ERBS). In
7


addition, ChIP-Seq also contains information on the density or strength of receptor
binding.

Based on such technology, numerous binding maps of important transcription factors
have been generated, including p53, Oct4 and Nanog (Loh, Wu et al. 2006; Wei, Wu
et al. 2006). For the estrogen receptor, at least five genome-wide maps of ERα
binding in MCF7 cells have been generated beside numerous other partial maps based
on chromosomes, promoters or custom loci (Cheung and Kraus, 2009). Based on the
different technique, 8,525 ERα binding sites were detected by ChIP-chip (Hurtado,

Holmes et al. 2008), 10,205 sites by ChIP-Seq (Welboren, van Driel et al. 2009) and
1,234 sites by ChIP-PET in estradiol-stimulated MCF7 cells (Lin, Vega et al. 2007).
Surprisingly, only a small portion of these binding sites were found in the proximal
promoter region of genes while the majority were distributed across the genome,
mostly in the region around 5-100 kb from the 5’- and 3’- ends of the adjacent
transcripts. Such binding characteristics were also observed in other transcription
factor and in other cell lines. This suggests that such transcription factors may
regulate transcription through long-range chromatin interactions.

1.3.2 Methods to study long range chromatin interactions

Chromosome Conformation Capture (3C) is the most widely used method to study
long range chromatin interactions across the genome (Dekker, Rippe et al. 2002). The
8


main concept behind the 3C technique is based on the “proximity ligation” concept of
the Nuclear Ligation Assay (Cullen, Kladde et al. 1993). In the 3C assay, the
chromatin is cross-linked with formaldehyde in the same way as in the ChIP assay
and digested by a restriction enzyme. The sticky ends of the fragments are ligated to
each other according to their spatial distances. Hence, fragments in close proximity
are more likely to ligate at a higher frequency. A classic example of this long-range
chromatin interaction is between the ß-globin locus and locus control regions in
mammalian cells (Tolhuis, Palstra et al. 2002).

Although 3C is a powerful technique, it does have several limitations (Fullwood and
Ruan 2009). First and most important of all, 3C experiments have high noise levels.
Consequently, 3C analysis relies on a set of control experiments to distinguish real
signals from noise, which makes 3C assay laborious and tedious (Dekker, Rippe et al.
2002). In addition, 3C methods are limited to single point interactions of previously

known or hypothesized interaction sites. In order to overcome these disadvantages,
several groups have developed new techniques based on the principles of 3C (Simonis,
Kooren et al. 2007), these include 3D (Hu, Kwon et al. 2008), Associated Chromatin
Trap (ACT) (Ling, Li et al. 2006), Chromosome Conformation Capture using Chip
(4C) (Simonis, Klous et al. 2006), Circular Chromosome Conformation Capture (also
called 4C) (Zhao, Tavoosidana et al. 2006), Open-ended Chromosome Conformation
Capture (Wurtele and Chartrand 2006) and Chromosome Conformation Capture
Carbon Copy (5C)

(Dostie, Richmond et al. 2006). Notably, 3D improves the
9


sensitivity of detection with DNA capture by using a specific biotinylated
oligonucleotide followed by DNA selection and ligation. This additional step detects
co-captured DNA fragments in a high-throughput and unbiased fashion, which in turn,
enhances the ability to detect long range chromatin interactions (Hu, Kwon et al.
2008). These new techniques provide new capabilities to detect long range chromatin
interactions, but are still constrained by their ability to provide a genome-wide view.
The development of highly efficient, low noise, genome-wide, and de novo method to
detect the long range interactions remains a challenge (Fullwood and Ruan 2009).

Recently a new strategy, chromatin interaction analysis by paired-end tag sequencing
(ChIA-PET), was designed to detect the global chromatin interactions (Fullwood, Liu
et al. 2009). In ChIA-PET, the long-range chromatin interactions are captured by
cross-linking with formaldehyde. The sonicated DNA-protein fragments are enriched
by ChIP process, followed by adding linkers and proximity ligation. The paired-end
tags are extracted, purified and sequenced. The sequencing results are mapped to the
reference genome to reveal the chromosome regions that are brought into close spatial
proximity through chromatin looping. This unbiased whole-genome approach has

greatly advanced our ability to study higher order organization of chromosomal
structures and functions (Fullwood, Liu et al. 2009).

10


1.3.3 Long range chromatin interactions of the estrogen receptor

Long range chromatin interactions mediated by the estrogen receptor of selected
genes has been reported by others. Using mainly 3C or 3C followed by ChIP, long
range chromatin interaction of distal ERα binding region and the proximal promoter
region has been reported for key E2 regulated genes such as TFF1 (Carroll, Liu et al.
2005; Pan, Wansa et al. 2008) and GREB1 (Deschenes, Bourdeau et al. 2007). Long
range chromatin interaction of the ERα binding site (144 kb upstream) with the
promoter of the NRIP-1 gene (Carroll, Liu et al. 2005), a ~ 6 kb upstream ERα
enhancer and the promoter of the CA12 gene (Barnett, Sheng et al. 2008) and a ~9 kb
upstream enhancer and the promoter of CTSD gene (Bretschneider, Sara et al. 2008)
were also reported to be mediated by ERα. Another recent example on ERα mediated
chromatin loop formed between the ERα binding site within the ERBB2 intron and
the ERBB2 promoter (Hurtado, Holmes et al. 2008), suggested that binding sites
within introns also contribute to long range chromatin interaction.

With the recently developed 3D assay, the first interchromosomal interaction between
the distal ERα binding sites of TFF1

(on chromosome 2)

and the proximal ERα

binding sites of GREB1 (on chromosome 21) were detected (Hu, Kwon et al. 2008).

Due to the dependence on ligand activated ERα, these recent data indicates a key role
of ERα in the mediation of long range chromatin interactions.

11


Using the ChIA-PET assay, a genome-wide chromatin interaction network mediated
by ERα was comprehensively mapped in MCF7 cells (Fullwood, Liu et al. 2009). In
all

by

1,451

ERα

mediated

intrachromosomal

and

15

ERα

mediated

interchromosomal long range interactions were reported. These interactions were
mostly anchored from distal ERα binding sites to gene promoters and suggest that

transcription regulation by distal ERα binding sites primarily function through long
range chromatin interaction. Data from the ERα ChIA-PET study has provided a
valuable starting point for future studies on the function of these distal enhancers and
the relevant genes that they target.

1.4 Aims and objectives of the study

Comparing the microassay results of the E2 regulation genes with the ChIP-Seq
database, we detect a serial of ERBSs around the possible E2 regulated genes. From
the ChIA-PET data, we are able to build a possible link between the distal ERBSs
with the target genes. This study narrows down to a specific E2 regulated gene, RET,
to investigate the estrogen regulation pattern and the corresponding ERα binding
affinity. More importantly, this study aims to investigate the possible long range
chromatin interactions around this target gene, which could be another solid example
to accomplish the ERα conducted chromatin interaction network. Besides, we also
intend to investigate whether the co-factors of ERα, like cJun, FoxA1, etc, have
functions in the ERα conducted chromatin looping.
12