Mod-06 Lec-21 Regulation of gene expression by steroid hormones


Welcome to this lecture on the regulation
of gene expression by steroid hormones. This is lecture number 21 in this course on
eukaryotic gene expression basics and benefits. On the last few classes, may be last 5 classes,
we have been discussing about how signaling molecules, which interact with membrane receptors,
ultimately activate or repressed transcription of specific genes. That is how signaling molecules interact in
cell surface receptor change or alter gene expression programs inside nucleus. We have been discussing at least 3 different
kinds of receptors. One of them called GPCR or G protein coupled
receptors which contain the 7 transmembrane domains and they what kind of signaling molecules
interact with these receptors and what kind of signaling cascade ultimately leads to activation
of gene expression. We also talked about receptor tyrosine kinases. How growth factors binds to growth factor
receptors, activates the receptors tyrosine kinases and how they through the map kinase
activity lead to ultimately activation or repression of target genes. In the last class, we discussed about signal
transducer and activator of transcription that is how starts or involved in the regulation
of gene expression by cytokines. There are number of other cells of a signaling
molecules and cells surface receptor pathways but we I gave you these 3 different pathways
as an example of how molecules interacting with cell surface receptors can ultimately
transudes signals through into the cell and activation of specific kinases can ultimately
lead to the activation of specific transcription factors. These results in the activation or repression
of gene expression and we also mentioned that there is lot of cross talk goes on between
these different signaling pathways and I gave you some of the examples. What we will do today? We will now go into the inside the cell and
discuss about those molecules which can diffuse through the cell membrane and enter inside
the cytoplasm and then bind to specific inter cellular receptors and how that results in
the activation or repression of specific target genes. One group of molecules which do this or steroid
hormones. So, what we will discuss in this class is
how steroid hormones, what are steroid hormones and how steroid hormones enter the cell and
bind to specific surfaces specific inter cellular receptors and activate or repress transcription
of specific target genes? So, this is what is the I have shown a schematically
here. So far, we have been discussing about molecules
which bind to specific receptors on the cell surface but today we are now going to talk
about molecules which can diffuse through the cell membrane, bind to specific receptors
and often when these molecules bind to these receptors, it results in the conformational
change. These modified receptors now often dimerize
and this dimerize then goes inside the nucleus, binds to specific promoter sequences and regulates
gene expression. So, we are going to use steroid hormones as
example of this group of molecules and ask the question how steroid hormones are able
to do this. That will be crux of today’s lecture. What are steroid hormones? Steroid hormones are steroids that act as
hormones. So, steroid hormones can be grouped into 5
groups based on the receptors through which they bind and these are known as glucocorticoids,
mineralocorticoids, androgens estrogens and progestogens and I just given the structure
of here cortisol is a glucocorticoid which is mainly made in the adrenal gland. Aldosterone is a mineralocorticoid again made
in the adrenal gland. Testosterone is a male sex hormone which is
made in the testes. Estradiol and progesterone are the female
sex hormones which are made in the ovaries. So, let us now and you can see now one type
get tell you the name those are all very important molecules. They control a number of very important cellular
processes and they all have more or less similar structure as you can see they are derived
from the cholesterol. So, let us now try to understand how what
are these molecule and how these molecules bring about their physiological effects? This is the structure of cortisol I have shown
here. They are derived from cholesterol and this
is a glucocorticoid which is synthesized from progesterone in the zona fasciculate of adrenal
cortex. The adrenal gland has 2 major regions called
the cortex and medulla. The glucocorticoids are made in the adrenal
cortex whereas the epinephrine and non-epinephrine which we discussed in the previous class which
are the molecules which interacts cell surface receptor, they are made from the adrenal medulla. Now, cortisol is involved in stress adaptation,
elevation of blood pressure, sodium uptake and numerous effects of immune system including
anti information. Many of the blocks which are available in
the market reduce information or glucocorticoids. So, glucocorticoids act as major anti-inflammatory
agents. All the steroids again, it is a mineralocorticoid. It is produced from progesterone again in
the zona glomerular region of the adrenal cortex. Again, it is involved in the raising of blood
pressure, adjusting regulation of fluid volume in the blood and also in the sodium ion uptake. So, it plays a very important role. Progesterone is a female sex hormone and it
is produced directly from the pregenenolone and secreted from the corpus luteum of the
ovary. It is responsible for changes associated with
the luteal phase of the menstrual cycle and is involved in the differentiation of mammary
glands, a very important hormone in the case of females. Testosterone is a male reproductive hormone
and it is an androgen. It synthesize in the testis and responsible
for all the secondary sexual male sexual characteristics. Estradiol is again a female hormone, again
produced from the ovary and is responsible for the female sexual characteristics. So, as you can see these 5 hormones are very
important. They have very important biological functions
and they are all synthesized from cholesterol. I have just shown here a slide that is taken
from this particular website which tells you how the glucocorticoids and the mineralocorticoids
namely the cortisol, corticosterone together constitute the glucocorticoids. Aldosterone and androstenedione they together
constitute the mineralocorticoids and how they are synthesized from cholesterol and
what are the various enzymes involved in this pathway? Ultimately, these molecules are synthesized. We will not go in details of how they are
made but just know that the glucocorticoids and mineralocorticoids, examples cortisol
and aldosterone. They are synthesized by the admiral gland
and they are all derived from cholesterol. Similarly, the gonadal hormones both Estradiol
and progesterone as well as testosterone, again cholesterol is percussive for by synthesis
and here is the pathway by which cholesterol is converted into the testosterone or estradiol. Need not remember the pathway at this stage
but just to tell you that they all are structurally very similar molecules but they have very
different physiological functions and they are produced in different regions of the body. These are natural steroid hormone that we
talk so far about. They are also what are called as synthetic
steroid hormones again play a very important role, has lot of biomedical applications. There are variants of structure that you have
just seen here and they have either more potent against or antagonistic action, mostly antagonistic
action. The antagonist also has very important effects. Glucocorticoid synthetic glucocorticoid for
example is called prednisone, dexamethasone, triamcinolone and so on and so forth. Synthetic mineralocorticoid fludrocortisones
these are all derivatives of the basic structure which I have shown in the previous slides. Many androgens in fact, they have very important
role especially those athletics for example. Athletes who want to have more muscles who
want to win races much faster; they take what are called as anabolic steroids which are
actually banned. Athletes are not supposed to take these anabolic
steroids, what is called as a dope test and if you test positive in this dope test, you
cannot participate in many of these competition. These anabolic steroids are nothing but synthetic
steroid hormones, especially synthetic androgens, oxandrolone, nandrolone and so on and so forth,
very important in the sports area. Estrogens again have a synthetic estrogen,
diethylstilbestrol called DES and you have synthetic progestins, non-ethindrone, medroxyprogesterone
acetate so on so forth. So, these steroid hormones are very important
biomedical applications because they are very portent molecules that alter the physiology. Now, some hormones have been around for a
long time. In fact, steroid hormones have been known
to exist since the early twenty century. However, it is only in the early in 1960s
and especially in late 1980s, the molecular mechanism of action of the steroid hormones
really became very clear. So, it was only until, it is not until early
1960s that the idea of specific hormone binding molecules in the target tissue of these hormones
began to emerge. So, till 1960, people did not know how these
hormones are working but then when bio chemistry in early 1960s or 70s, the biochemistry ruled
the world in the biology and when the biochemistry starts grinding the tissues and started looking
what kind of protein molecules bind to this. Steroid hormones, it should become very clear
tissues which are response to this hormone molecules actually contain proteins which
bind to this hormones steroid hormones. So, the concepts that these hormones may be
acting by actually binding to specific receptors began to emerge by bio chemical studies that
carry carried out in the early 60s. The analysis of the steroid hormone receptors
had relied largely on bio chemical techniques as I said the one of the major methods by
which people use to study steroid hormones, the late 1960s and early 1970s is you label,
use a radio label steroid hormone and then you see how the radio label hormone goes and
binds to protein that results and then try to monitor to the kinetics of binding and
then see where this receptor is localize and so on and so forth. So, primarily bio chemistry contributed a
lot to understanding the molecules of the protein molecules which actually bind to the
steroid molecules in the early 60s but it is only after the genes encoding these receptors
were cloned, it became possible to carry out detailed studies of various functional domains
of the receptor. So, the best that bio chemist could do is
to see what kind of molecules bind and what is the affinity of this steroid hormones to
these molecules and with a great difficulty they could actually purify some of this receptor
molecules. In late 70s some of these proteins or the
receptor molecules could also be sequence, so the partial amino acid sequence of some
of these steroid hormone receptors were available but complete characterization was not possible
using this bio chemistry alone but the late 70s and the early 80s started was the drawn
of molecular biology. It became to clone genes; it became to identify
the amino acid sequence of proteins using the genes sequence so on so forth. Once restriction enzyme is cloning, technique
became available that made a very major role in understanding the function structural and
function of steroid hormone receptors. So, what I am going to do is go through take
you through a brief historical perspective of what kind of people are actually contributed
a lot to the understanding of the steroid hormone receptors structure and function and
what is the historical perspective of this filed. That is what we will discuss in next few slides. Now, as I said bio chemistry played a major
role in understanding the structural function of steroid hormone receptors. For example, in the 1968 some of the studies
actually led where you take a tissue, homogenize a tissue and then do a different gentrification
by you can separate nuclei and you can separate a cytoplasm and then with they took this different
faction and see where is the protein which is able to bind to this steroid hormone is
present. Based on such studies, people actually showed
that when there is no hormone, this binding protein is actually present in the cytoplasmic
fraction but the movement we have hormone, this activity sifted to the nucleus. So, this kind of bio chemistry started giving
an idea of how these molecules actually act or how these molecules are able to bring about
physiological response and they clearly told that these molecules actually enter inside
the cell and binding to specific intracellular proteins. Again, once people started purifying using
these kinds of technical localization studies and again using bio chemical purification
techniques, people have started purifying this receptor protein from some of this tissue. Once you got a purified receptor, you could
actually do a partial styptic digestion of this receptor or find out N terminal acid
sequences or internal peptide sequencing and based on these peptides, as well as the purified
receptor, you can immunize either mice or rabbits and you can generate antibodies. So, once you have antibodies which are specific
for this kinds of steroid hormone receptors, then people use this antibodies to ask the
question where exactly this receptor are present. For example, if you have a specific tissue
which has a binding protein for that particular steroid hormone, you do what is called immuno-fortis
technique where you order receptor anti-antibody to specific that particular receptor. Then we have what is called the second antibody
which is always conjugated to florescent molecule, it could be what has called florescent isothicyanate
or and so many other modern dyes which under florescent microscope either give a blue colour,
green colour or a red colour. So, using this kind of immuno-fluorescence
technique people and using receptor specific antibodies, people could actually demonstrate
that these receptors when there is hormone inside the cell they are actually present
inside the nucleus. So, picture began to emerge that these steroid
hormones basically act by binding receptor molecules present inside the cytoplasm and
once the hormone binds the receptor, this receptor is going to the nucleus and probably
it is activating the transcription of specific genes. That is how these steroid hormones are able
to bring out specific biological effects. This is the paradigm that began to emerge,
so many of this bio chemical studies that was carried out in the late 1960s. So, this is what I have summarized. What I just told you here, it is generally
thought that unoccupied steroid receptors can exist in the cytoplasm while the occupied
receptors act in the nucleus on specific target DNA sequences but these are all again not
much experimental evidences available, it is mostly conjunctional. So, when bound to the hormones cytoplasm,
hormone receptors move to the nucleus and the purified receptors probably are going
and binding to specific DNA sequences. Some of the key experiments which were done
again using bio chemistry and little bit of molecular biology because as I said the late
70s and 1980s is the one that was the don of molecular biology. A number of molecular biology techniques became
available and people started using, especially bio chemist who were actually doing protein
purification and protein characterize and technique now realize that they could answer
many of this question using many of these molecular biology techniques by looking at
the transcription by looking at the genes and so on and so forth. So, what for example Keith Yamamoto’s group
actually did in 1983. They published paper in cell in 1983, sequence
specific binding of glucocorticoid receptors in MTVDNA at sites within and upstream of
the transcribed region. So, that is virus called mouse mammary tumor
virus and this virus response very well. The trans for the transcription of this virus,
you require glucocorticoids. So, people like Yamamoto started asking question,
how does this mouse mammary tumor virus promoter is getting activated by glucocorticoids because
glucocorticoids is producing in our body and here is a mammalian virus and the transcription
of this virus is actually getting activated by a hormone which is produced by our body. So, what they did? They actually took what is called the long
terminal region long terminal repeat or LTA of these particular MMTB virus or MTV virus
and then ask the question where exactly does the glucocorticoid receptor binds because
by this time, it is very clear. All the effects of glucocorticoids are probably
modulated to the glucocorticoid receptor. So, if the glucocorticoid is activating the
expression of this mouse mammary tumor virus, people ask the question. The glucocorticoid receptor is must be doing
something to the virus and the first question they ask this does it go and bind to specific
DNA sequences within the mouse mammary virus tumor DNA. In fact, actually showed that there are about
5 regions within the MTVDNA, they are specifically bound by purified glucocorticoid receptor
and one result upstream of the transcription start site and others are distributed within
the transcribed sequences between 4 to 8KB from the initiation site. So, for the first time studies like this are
actually shown that here is a receptor mammalian receptor that is actually binding to some
specific sequences within this viral genome and this binding is probably responsible for
activation of expression of the viral genes. So, such studies with purified steroid hormone
receptors demonstrated that they are likely to be sequence specific DNA binding proteins
and they are likely to be transcription factors. So, this is what emerged for many of these
bio chemical studies. So, classical purificate, classical ligand,
receptor interaction studies, understanding their kinetics of binding and affinity of
this ligands to this receptors as well as bio chemical purification techniques followed
by several localization using immuno-fluorescence as well as and more late 70s and early 80s
using some of the molecular biology techniques, it became very clear that one of the major
mechanisms by which these steroid hormones are acting is by binding to the steroid hormone
receptors and these steroid hormone receptors are then going inside the nucleus and bind
to specific regions within the DNA sequences. That is how they are able to bring out specific
physiological effects. So, this is the paradigm that emerged before
the molecular biology era started. The most important phase in the study and
understanding of the steroid hormone receptor function began with the cloning of steroid
hormone receptors. So, I am going to give you a brief historical
prospective of how this steroid hormone receptors were cloned. Now, studies from a number of laboratories
such as those headed by Elwood Jensen, Bert O’Malley, Keith Yamamoto and many others,
I am not going to quote everybody who worked in those areas these are some of the very
important people who made very important contributions to the understanding of steroid hormone receptor
functions. All these studies actually led to the hypothesis
that activated steroid hormone receptors go and bind to specific DNA sequences in the
nuclear of the target cells and this is what induces the transcription of these genes leading
to specific production of specific proteins. So, all the bio chemicals studies and immuno-localization
studies and typical receptor ligand interaction studies, all these things pointed out that
when you add a hormone to this steroid hormone to the cell, it is entering the cell, binding
to this inter cellular receptors. Then it goes inside the nucleus and this binding
of the receptor to specific DNA sequences results in the synthesis of specific proteins
and that is how the steroid hormones are bringing about their physiological responses. Now, by the end of 1980s when techniques for
cDNA library screening and cloning and sequencing DNAs became a routine, the first receptors
cDNA encoding the glucocorticoid receptor was cloned by Ronald and Evans in the Salk
Institute in La Jolla California united states of America. So, 1980s as I told bond was the golden era
of biology because the molecular biology techniques became available. People realized that you can actually purify
messenger harness for a specific protein, encoding this specific protein and you can
derive a cDNA of those and you can actually clone those DNAs. So, from millions and millions hundreds and
thousands of genes which are available, you can actually pull out a specific gene or specific
cDNA coding for a particular protein. So, once the creation of cDNA libraries and
how to screen these cDNA libraries and how to isolate the specific cDNA became available,
people began to understand can we clone a cDNA that clones for or can we clone messenger
RNA that actually syntheses this steroid hormone receptors. The first one was done by Ron Evans Salk institute
in the late 90s the early 1980s. Once they receptors glucocorticoid receptor
was cloned, this was soon followed by cloning of the cDNA encoding for estrogen, progesterone,
androgen so on and so forth cloned in subsequent years. So, I am going to spend some to tell you how
actually all these things were done because these are land mark papers in the area of
steroid hormone receptor structure and function. So, let us now see what exactly they did. So, the first paper on the characterization
of the cDNA encoding the glucocorticoid receptor was published in journal nature in 1985-1986
by Ronald Evans Salk Institute and his entirely primary structure and expression of a functional
Glucocorticoid receptors cDNA. These how would considered as a land mark
paper in the area of steroid hormone receptors structure and function because this marked
the beginning of a new era in the bio chemistry and this paved way are this probably is primarily
responsible for our current understanding of the steroid hormone receptors structural
function. So, what this group actually did is to demonstrate
that there are 2 forms of glucocorticoid receptors cDNA’s. So, what basically they did? They took the RNA isolating from a tissue
which express a glucocorticoid receptor, converted them into cDNA by using reverse transcription
and so on and then took the cDNA’s of all these messengers or sensor in the tissue and
then put them in the page vector and then made a library. So, I have basically made a cDNA library and
then using either antibody specific for glucocorticoid receptor or using oligonucleotides which specifically
or digging the oligonucleotides that corresponds to the specific amino acid sequences of this
glucocorticoid receptor which is available at that time because the purified receptor
could be cleaved and the partial amino acids sequence could be identified for some of the
peptides using either oligonucleotides that corresponds to those peptide sequence or by
using antibodies rising purified glucocorticoid receptor. You basically, screen these libraries and
ask the question which phase clone are for the cDNA that actually cores for the glucocorticoid
receptor. So, by cDNA screening, cDNA library screening
they have actually identified 2 clones and one of them cloned for a glucocorticoid receptor
of 777 amino acid and they called it as glucocorticoid receptor alpha. Another cloned for a 742 amino acid protein
which they called as a glucocorticoid receptor beta and you can see this is the correct knowledge
of glucocorticoid receptor structure and the foundation for this was actually laid in the
year 1985-86 by Ronald Evans group. We now know that all these amino acids are
encoded from about 9 hexons. This is the hallmark from 1 to 9 here and
today, we know that the glucocorticoid receptor structures actually consists of what is called
as a N terminal domain and it consists of a DNA binding domain which actually comes
from hexon 3 and 4 and consists of what is called as ligand binding domain to which the
glucocorticoid goes and binds. So, the alpha is after this whereas the beta
is truncated here. Now, in the same year from the same group,
it also became another important land mark. Paper was published where it was reported
that the domain structure of the glucocorticoid receptor and its relationship to the v-erb-A
gene and its relation to v-erb-A oncogene product. Now, I can see here the importance of molecular
biology. Now, the bio chemical purification studies
could only at the most partial amino acid sequence, all they could raise antibodies
but nobody could obtain the full length amino acid sequence of glucocorticoid receptor using
conventional bio chemical techniques because it is impossible for you to sequence the entire. How many amino acids? Almost 777 amino acids, you cannot do typical
amino acids sequence of these proteins this 2 v-erb recurring a draw but once the cDNA
sequences are available from the cDNA sequence; you can actually deduce the amino acid sequence
and based on the amino acids sequence, people ask the question what kind of a protein is
this? What kind of functional domains are actually
present in this glucocorticoid receptor? Based on such studies in the subsequent paper
they actually shown that this glucocorticoid receptor some of the domains actually resemble
to that of an alkaline protein called v-erb-A. Now, what is this v-erb-A? The v-erb-A is an oncogene product of a virus
called avian erythroblastosis virus or AEV. So, these are viruses which when we infect,
it actually causes cancer and one of the major proteins which is responsible for causing
cancer in this virus is a protein called v-erb-A. What the studies of this group actually shown
is that the structure of the glucocorticoid receptor very much resembles the structure
of this oncoprotein. So, you can see people who have been studying
steroid hormone, suddenly realize that there is some relationship between steroid hormone
receptors and cancer because here is a viral protein which is causing cancer and the structure
of protein is resembles very much that that of that glucocorticoid receptor. So, people asked the question, what is the
link? So, they predicted in this paper that the
oncogencity of this virus may result in part from the inappropriate activity of a truncated
steroid receptor or a regulatory molecule encoded by v-erb-A.
So, the movement they found that structurally these two proteins are related. People realised that the mechanism by which
this oncoprotein is acting causing cancer, may be similar may be because of inappropriate
signaling. So, they suggested that a mechanism by which
transcription factors may facilitate transformation. So, these kinds of studies clearly showed
that transcription factors may be involved in similar transformation leading to cancer
and they also identified another short region in the human glucocorticoid receptor which
had very high degree of homology to certain proteins which are involved in the regulation
of development like The Drosophila homeotic proteins encoded by Antennapedia and fushi
tarazu etcetera. Now, we will come back to this transcription
factor regulation in development sequence places but what I am trying to tell you is
that once the cDNA for the glucocorticoid receptor was cloned and the domains structure
was analyzed, it became very clear that this is like to be a transcription factor. There is something look like a DNA binding
domain and it also had a homology to very important viral protein which causes cancer
and people realized that the mechanism by which this virus are causing cancer may be
because of inappropriate activation of some of the genes which are actually activated
by steroid hormone receptors. Now, they soon realized that the subsequent
here 2 groups are actually published a paper where they actually demonstrate the v-erb-A
gene is nothing but a thyroid hormone receptor. So, the link between cancer and hormones became
firmly established when they realized that one of the viral oncoprotein which very much
resembles that of a glucocorticoid receptor actually course for a thyroid hormone receptors. I can see as I have just highlighted some
of the important findings from this paper where this group, again from Ronald Evans
lab actually showed the c-erb-A gene encodes a thyroid hormone receptor. What they showed is that the cDNA sequence
of homology and c-erb human the c-erb-A the cellular counterpart of the viral oncogene,
v-erb-A indicates that the protein encoded by the gene is related to steroid hormone
receptors and binding studies with the protein show it to be a receptor for thyroid hormone. So, they took the gene encoding for the thyroid
hormone receptor, did what is called as invitro transcription and translation and that is
you take the RNA, translate in the invitro and this invitro translate protein was able
to bind to thyroid hormone. So, here is a viral oncoprotein involved in
cancer that seems to thyroid hormone clearly saying that there is a link between the hormone
signaling and cancer. Again, here in the same you can see these
two papers are published in the same issue of nature back to back. This is from Vennstorm lab in Europe where
again it is showed that hormone binding and localization of the c-erb-A protein suggest
that it is a receptor for thyroid hormone, a nuclear protein that binds to DNA and activates
the transcription. So, the product of viral oncogene is defective
in binding to the hormone but still localised to the nucleus. So, you can see here is a viral oncoprotein
which binds to a DNA but it is not bind to thyroid hormone clearly indicating that if
we have receptors which have defective in ligand binding or which are hormones binding
and such receptors may cause inappropriate signal transition pathways activate inappropriate
oncogenes. This is what can lead to ultimately cancer. So, these kinds of studies established a link
between viral virus induced cancers or a viral oncogene products and hormone signaling pathways. So, the cloning of glucocorticoids receptors
are in the same time Ron Evans group clones glucocorticoid receptor. A number of other laboratory started cloning
other receptors because they all had either antibodies of specific receptors or they had
the partial peptides sequence amino acids sequence or salten peptides of this various
receptors. Pears Chambo group in France for example took
the antibodies against the glucocorticoid estrogen receptor as well as they made ologo
neutralize against peptide amino acid of specific peptides of the estrogen receptor and they
cloned the estrogen receptors cDNA. So, they reported the cloning of the cDNA
of estrogen receptors in the breast cancer cell line MCF-7 and expression of the ER cDNA
in hela cells produces protein that has a same relative molecular mass and binds oestradiol
with the same affinity as MCF-7 ER. So, it became very clear that protein which
is actually produced from this cloned gene is more over the same as the estrogen receptor
that is expressed in a well-known breast cancer cell line. So, this they again they also reported that
there is extensive homology between the estrogen receptor and the erb A erb-A protein of the
oncogenic avian erythrocytosis virus. So, all these became clear that whether you
clone glucocorticoid receptor, whether it is an estrogen receptor, they all seem to
have similar structure and the structure is very similar to that of an oncoprotein encoded
by oncovirus or a tumor virus. So, all these studies clearly led to the proposal
or hypothesis that there exists a super family of oncogenic hormone receptors. So, there are certain oncoproteins which are
highly homologous to the hormone receptors and this super family of receptors may be
actually involved in viral transformation. So, both Chambon as well as Roan Evan group,
actually proposed that these human steroid receptors as well as some of this viral oncoproteins
like erb-A may actually constitute a super family of enhancer binding proteins and they
may actually cause cancer. Once the glucocorticoid receptor cDNA and
estrogen receptor cDNA was cloned, subsequently number of other steroid hormone receptors
was cloned. For example, within 1960s 1968 for example
the chicken progesterone receptors cDNA was cloned as well as the human androgen receptor
cDNA was cloned. So, once all these cDNA for this steroid hormone
receptors was cloned it became very clear that they all encoded super family of hormone
receptors. This I am just putting a slide which I have
already explained here. What was the strategy that was employed for
cloning of all these genes including steroid hormone receptors? The strategy is to make a cDNA library that
is you isolate messenger RNA from all these tissues which are expressing these specific
receptors and then you convert all this messenger RNA into cDNA and then you clone this cDNA
into phage vector and make a phage library or a phage DNA library. Then take this phage cDNA library and you
either probe them with antibodies against specific receptors which have been purified
from various cell types. You take these antibodies against the purified
receptors or make oligonucleotides degenerate oligonucleotides corresponding to the amino
acid sequence of specific receptors and using either any labeled oligonucleotides or use
antibodies, you can screen this phage libraries phage reading libraries. That is what they can isolate this cDNA clones
and then you sequence this cDNA clone and reduce an amino acid from cDNA sequence and
see what kind of a protein that they are coding for. So, this was a strategy that was used for
isolating the various steroid hormone receptors in the early 80s and late 1980s. So, once all these studies were done, you
have the receptor of glucocorticoid receptor, you had the receptor of estrogen receptor,
thyroid hormone receptor so on and so forth. It became clear from all these studies that
all these receptors had some common structural domain. For example, all these receptors have what
is called as the ligand binding domain which probably binds the hormone. It is called as the hormone binding domain
or the ligand binding domain. The receptors also has what is called as DNA
binding domain in the middle and this probably responsive for binding specific sequences
and it also what is called as amino terminal hyper variable region which was not that will
consult between various receptors that then go so on and so forth. So, some of the three major functional domains
of this receptors were a ligand binding domain, the DNA binding domain and hyper variable
amino terminal domain. So, this is for the major domains which are
present in all these receptors. Today, we now know that there are much more
detailed information available on the various domains of the steroid hormone receptors. For example, we now know that this amino terminal
region which is actually shown as A slash B actually contains what is called as an activation
function 1. This is actually responsible for ligand independent
activation of steroid hormone receptors and we also know that the region known as C, actually
course for a DNA binding domain and this DNA binding domains consist of 2 zinc fingers. We have discussed about zinc fingers in the
previous classes when we talk about the structure and function of various DNA binding proteins
and this steroid hormone receptors contains 2C2H2 type of zinc fingers and we will look
at this structure in a few more minutes. It also be very clear people started to looking
at the mechanism by which this DNA binding domain acts while discuss in more detail in
a few more minutes and this DNA binding domain what is called as the P box and what is called
as a dimerisation region and plays a very important role in DNA binding function of
the steroid hormone receptor. As I said all these steroid hormone receptors
are present in the cytoplasm when there is no hormone, they go into the nucleus in the
presence of hormone. So, they contain what has called as nuclear
localization signal and this nuclear localization signal is massed when there is no hormone
once the hormone binds its induced conformational change. So, the nuclear localization signal gets exposed. Therefore, receptor goes inside the nucleus. Now, as was ligand binding domain, the ligand
binding domain has two important functions. It has a ligand dependent activation function
that means there is an activation function 2, whereas the one in the amino epidemic terminal
referred as activation function 1. When ligand binds, this activation function
is activated and now this in the presence of ligand, the receptors able to interact
with the general transcription machinery and the RNA polymerize resulting in the activation
of transcription. The domain also in the absence of ligand in
certain receptor can act as a repressor. So, the cloning what of this cDNA’s of these
receptors paved way for a very detailed understanding of the various functional domains of these
various steroid hormone receptors. Now, once all these receptors were cloned,
there also many it also became clear that the structure of this steroid hormone receptors
made homology to other non-steroid receptors, for example thyroid hormone receptors. Thyroid hormone is not a steroid hormone but
receptor for thyroid hormone was very homologous to then of the glucocorticoid receptor or
estrogen receptor. So, people soon realized that there exists
a nuclear receptor super family and this nuclear receptor super family contains not only steroid
hormone receptors but also contains other non-steroid hormone receptors or other hormone
receptors and which are not steroids. So, all these based on the structural homology
were grouped under one nuclear receptor super family. So, by sequencing cDNA’s of various hormone
receptors and deducing the amino acids sequences, all this were grouped under a single nuclear
receptor super family. Two broad categories of this receptors exists
in this one are called as type 1 receptors, another called as type 2 receptors. The type 1 receptors, basically consists of
steroid hormone receptors. They undergo nuclear translocation of the
ligand activation and bind as homodimers to inverted repeat DNA half sites refer to as
the hormone response elements. So, the DNA sequences to which the steroid
hormone receptor binds or HRE or Hormone Response Elements. Glucocorticoid well bind to glucocorticoid
response element, estrogen will binds to estrogen response elements, thyroid hormone bind to
thyroid hormone response element so on and so forth. So, in general they are known as hormone response
elements. So, the example for the type 1 receptor are
receptors activated by steroid ligands such as glucocorticoid receptor, mineralocorticoid
receptor, estrogen receptor, progesterone receptor and androgen receptor. So, all these steroid hormone receptors are
classified as type 1 receptors because usually they stay in cytoplasm in the absence of the
hormone and once the hormone binds the conformational change, then going to the nucleus and often
bind what is called as inverted repeated sequences. We will come to that in a minute whereas the
type 2 receptors, they often are retained in the target cell nucleus regardless of the
presence of the ligand. So, unlike classic hormone ligand receptors
which are present in cytoplasm in a hormone and goes into the nucleus only when there
you have hormone. The type 2 receptors are already present into
the nucleus, irrespective of whether the hormone is present in the hormone or not. So, they are nuclear receptors. Whereas, steroid hormone receptor cytosolic
in nature in the absence of hormone and nuclear in nature in the presence of hormone and the
type 2 receptor is always nuclear in the nature The function of ligand is actually to unfold
or to activate the transcription activation function of the type 2 receptor. So, in the absence of hormone, although the
receptor is bound to the DNA, it does not activate transcription but both the hormones
binds; it induces a conformation change. Therefore, the receptor can activate transcription. Examples for type 2 receptors are thyroid
hormone, retinoid acid, vitamin D so on and so forth. We will discuss type 2 receptors in the next
class but today we will confine ourselves to only for the type 1 receptors which basically
comprise of the steroid hormone receptors. Now, so it became very clear that these steroid
hormone receptors act or bring about their physiological effects like primarily binding
to what are called as the hormone response elements which are present on the promoter
regions of various genes. So, once the hormone binds, the intercellular
receptors now functions as transcription factors. They go and bind to the hormone response elements
in the promoter regions of various genes and this is how they act as transcription of various
genes. So, all the genes which are the target called
glucocorticoid hormone. They invariably contain a hormone response
element in the promoter region and that is how transcription of these genes are getting
activated in presence of those hormones. So, if the target gene needs to be activated
by glucocorticoid, then you should contain glucocorticoid response element. If the product gene is to be activated by
estrogen, it should contain an estrogen response elements and so on so forth. So, by having specific hormone response elements
in the promoters of these target genes, these hormones by through their receptors are able
to bind this in the sequences and bring about transcription activation. So, this became the general mechanism by which
steroid hormones bring about transcription activation. Now, one of the important experimental techniques
that actually played a way for understanding the structural function of this nuclear receptors
and the steroid hormone receptor is assay called as cis-trans cotransfection assay. It is because of this assay, it became possible
to identify the various functional domains of these steroid hormone receptors. I have already discussed this cis-trans cotransfection
assay in one of the earlier classes, especially the introductory classes of this course but
I do not mind repeating again. Basically, what this actually means is that
this is a transient transfection based hormone response element reporter assay developed
again in the laboratory of Ronald Evans at salt institute and it became the tool of choice
for investigating the mechanism by which nuclear receptors regulate their target of genes. So, this is a very important assay and because
of this assay that people could identify the various functional domains steroid hormone
receptors. What is this in this assay? A cDNA encoding the receptor or part of the
receptor is transfected into a suitable cell line along with a reporter gene usually can
be luciferase gene link to a promoter controlled by one or more of the hormone response element
specific for the receptor being studied. So, basically what to do in this assay is
that you take the cDNA that codes for, let me say I want to see whether glucocorticoid
receptor actually activated from a promoter or not or whether a promoter actually contains
a glucocorticoid response element or not. So, what I do I take the promoter sequence
or I take the sequence which suspected in the glucocorticoid response element and link
it to a luciferase gene which is a reporter gene, it can also be or can be transferase,
there are number of such reporter genes. So, take the luciferase gene, put it down
stream of this suspected promoter which is response glucocorticoid and put this plasmin
inside the cell. This is called as the cis factor. Now, you take another plasmin which contains
let us say for example a well-known viral LTR viral promoter which is known to be expressed
by host transcription factor in a number of cell line. So, clone the receptor cell for glucocorticoid
receptor under this promoter, so that when you put this plasmin inside the cell, the
transcription factors present inside the cell go and bind to the promoter and they express
the receptor. Once the receptor is expressed, the receptor
will now go on and bind to the cis vector, the promoter region of cis vector and now
if we add ligand, it will activate the transcription. So, if the cis plasmin contains the sequence
for binding of the hormone receptor, then when the receptor is expressed from transplasmade,
it will go and bind to luciferase vector and induce the express the luciferase gene. You can then you can measure in the luciferase
activity and then you can now demonstrate that yes, this receptor can actually activate
transcription activation from a specific region. So, by using this assay not only you can identify
hormone response elements in promoter region of any gene, you can also bisect out what
region of the receptor is important. For example, you can make mutations by DNA
binding domain and ask the question can it go and bind to DNA? Can it now activate transcription or you can
change amino acid sequences within the DNA binding question and ask the question how
is this specific domain or you can switch the ligand binding domain. For example, since I have DNA for estrogen
receptor, have a cDNA glucocorticoid receptor, I can take the ligand binding domain of the
glucocorticoid receptor and fuse it to estrogen receptor. Now, if I take the primary receptor and put
inside the cell and since the glucocorticoid receptor, now has an estrogen receptor cDNA,
now it will activate transcription from glucocorticoid response element in response to estrogen but
not in response to glucocorticoid. These kinds of experiment done using this
assay clearly indicate that these steroid hormone receptors have a modulus structure. You can take the DNA binding group of glucocorticoid
receptor and put it on estrogen receptor or you can take then DNA binding of estrogen
receptor, put it inside glucocorticoid repeat and the deal is they start activating from
each other response element that is the glucocorticoid response receptor contains estrogen response
receptor DNA binding domain will now start activating transcription in estrogen response
element or you can do other way also. You can take ligand glucocorticoid receptor
binding domain or the ligand binding domain glucocorticoid receptor and switch it to that
of the estrogen receptor. Now, they start activating transcription with
each other ligand. So, these kinds of cis cotransfection assays,
they would say for characterization of the DNA binding domain as well as ligand binding
domain of the various steroid hormone receptors. So, such studies actually demonstrated that
steroid hormone receptors actually bind to hormone response elements and this actually
led to the characterization of the various steroid hormone response elements. For example, hormone such as progesterone,
androgen, glucocorticoid and mineralocorticoids, they all seem to bind to a specific the same
kind of response element containing AGAACA separate and TGTTCT separated by three nucleotides. This is actually called as an inverted repeat. You can see AGAACA and the opposite stands
as TGTTCT, so AGAACA and the opposite stand also AGAACA and therefore is called inverted
repeat. So, AGAACATGTTCT and the opposite stand again
lead as AGAACA, so how this same AGAACA sequence on the two sides of the double DNA. So, these are called as palindromes or inverted
repeat sequences. So, many of these steroid hormone receptors
bind to the repeat sequences, it should become one of the hallmarks of hormone response element. Interestingly, all these four receptors progesterone,
androgen receptor, glucocorticoid, mineralocorticoid, they all bind to a similar sequence which
consists of what are called as two half sides. The AGAACA is one half side, TGTTCT is another
half side and these two half sides are separated by three nucleotides, it can be any nucleotides. So, when you have this kind of sequence in
the promoter region of gene, you can say this is lightly to be activated by any one of these
hormones. Similarly, if you take estrogen receptor,
estrogen receptor actually binds to a sequence called AGGTCA, 3 nucleotides TGACCT. So, you can see the only difference between
estrogen receptor or estrogen response element and a glucocorticoid response element is two
nucleotides. So, if the sequence is AGAACA, it becomes
glucocorticoid element instead of AGAACA. If it is AGGTCA, it becomes estrogen response
element. So, you can see the physiological effects
of estrogen is quite different from physiological effects of glucocorticoids but if you see
at the molecular level, the only these two of the ways very fine difference that distinguishes
between the estrogen receptors and progesterone receptor target genes. So, they target gene promoter target genes,
the promoter contains AGAACATGTTCT becomes responsible to glucocorticoid. If the same sequence instead of the 2A replaces
such by AGT, it now become a response for estrogen receptors. So, this is a very fine difference exists
at the molecular level between genes that are responsible for glucocorticoid and the
genes that are responsible to estrogen and very fantastic experiments that actually done
at the future characterized DNA binding through steroid hormone receptors. So, it became clear that these all these steroid
hormone receptors whether it is GRMRPRE RRAR, they all contain two zinc finger domains and
this zinc is coordinated by cystine-rich. So, they contain the C4 type of zinc fingers
and it has been very well shown that the DNA binding domain actually contains two very
important regions called as P box and D box. The D box refers to dimerise box or a distil
box and P box is the proximal box and many number of people actually could do mutation
within this the DNA binding domain and ask the question, what are the amino acids residues
which are important for DNA binding. For example, if you now mutate this particular
A adenine in this DNA binding alinine in this region. If you mutate that a quanine, now this receptor
can no longer dimerize. So, if you mutate the IM residue that of in
this amino acid position 458 in the glucocorticoid receptor to the glucocorticoid receptor cannot
dimerize. So, can now just go back and tell you here,
since the hormone response amino consists of palindromes that means you have two half
sides AGAACA1 on one stand and AGAC on other stand. Therefore, the steroid hormone receptors in
order to activate transcription and they go on and bind as dimers. So, one hormone recognizes its one half sides
on one stand and the other hormone recognizes on the other stand. In order to follow to bring together, we need
a dimerisation domain. There is a dimerisation interface domain and
there is also dimerisation interface in the ligand binding domain of all these steroid
hormone receptors. So, using this kind of cis-trans cotransfection
assay people have to identify what are the important amino acid residues that play very
important role in DNA binding. Like I said in the case of glucocorticoid
receptor, one mutation actually can abolish the dimerisation function of the receptor. A very important experiment that was done
in the 1989 in the year 90 by the two groups, one headed by Gordon Ringold lab as well as
Ron Evans lab in the Salk Institute, they actually demonstrated by making specific mutation
within this DNA binding domain, you can actually change this specificity of one receptor to
other. For example, in this paper published in cell
where they actually showed two amino acids within the knuckle of the first zinc finger
specify DNA response element activation by the glucocorticoid receptor. What they actually showed in this paper is
that the ability of glucocorticoid and estrogen receptors were discriminated between the close
related response element results in two amino acids located between the two cysteines in
the C-terminal half of the first finger. I have told you glucocorticoid response element
is AGAAC half side whereas the estrogen receptor instead of AGAACA it is AGGTCA. So, only two nucleotides are different. What is paper actually include is that if
you change two amino acids in the DNA binding domain of the glucocorticoid receptor is that
to recognize that glucocorticoid receptor is now recognized to that receptor. A similar paper was published in the same
issue of cell and this is the amino acid by again from the Ron Evans group. Again the same issue of cell where actually
showed as that simply change in rising residue to glutamate in the base of the first zinc
finger in the P box, you can now convert glucocorticoid receptor to that of estrogen receptor with
respect to DNA binding specific is concerned. So, if we simply mutate one amino acid glazing
to glutamate instead of this receptor recognize glucocorticoid response element, it will go
and recognize estrogen response element. So, you can see we have a number of steroid
hormone receptor from the gross structure is the same. They all contain a zinc finger domain but
minute amino acid differences between these receptors actually distinguish whether this
was going to bind glucocorticoid response elements or whether go and bind to a estrogen
response element. So, palindromic and all the steroid hormone
response elements exists as palindromic sequences and the receptor actually goes and binds as
a dimer as shown here. One hormoner comes and binds one half side
and another hormoner comes and binds another half side. That is how there is a bind dimer to the target
DNA sequence and activates transcription and as I mentioned here the glucocorticoid response
element and estrogen response element differ from each other from two nucleotides if is
AGAACA half side is a glucocorticoid response element if the AGGTCA half side it becomes
estrogen response element. If you just make one amino acid mutation within
a DNA binding domain of glucocorticoid receptor instead of recognizing this sequence, now
the glucocorticoid receptor recognize this sequence. So, you can see how beautifully the nature
has evolved target genes specific of this steroid hormone receptors. Now, one can ask question, you have glucocorticoid
receptor, mineralocorticoid receptor and androgen receptor, they are all recognised the same
sequences, then how is this specifically brought about because the mineralocorticoid physiological
effects are different and glucocorticoid physiological effects are different. Androgen receptor physiological effects are
different but they all are binding the same sequence. So, how is that specific stage brought about? How specific physiological response are brought
about by these different receptor, despite the fact that DNA binding by same sequence. So, the answer is although many receptors
like GR, MR and AR, they all recognize with the same sequence, the specificity achieved
by what kind of receptors actually present. For example, a tissue which is responsible
to glucocorticoids may only have glucocorticoid tissue which is responsible. Glucocorticoids may have only glucocorticoid. It may not have a mineralocorticoid receptor,
therefore it become responsible for glucocorticoid receptor. So, by expressing specific receptors in this
target types, they can achieve specific target gene specific receptor. So, very rarely you will find both the PR
and GR may be expressed in the same type of cells. Even they are expressed in the same types
of cells; the co-activators may require for the activation may be present in one receptor
but not the other. We discuss this later in the lectures series. So, although the target genes specifically
may be similar depending up on the cell type and tissue specific expression of the receptors
as well as the different coordinated requirements like different gene may be activated and different
receptor may be activated in different cells. So, these kinds of experiments have been actually
done again by very nice experiment where you can actually demonstrate this kind of specificities
by actually, for example in the estrogen receptor, how do they demonstrate the receptor can actually
activate from the estrogen response element but not from glucocorticoid response element
in the same cell type. You can actually show you can take the estrogen
receptor cDNA and transfer this in an expression that and transfer this expression into the
cells of your interest when the glucocorticoid estrogen receptor is expressed. Now, if we had an estrogen hormone into this
cell, it will now activate only those genes which can estrogens response element but it
will not activate transcription of those genes containing a glucocorticoid response element. Indicating that these receptors binding into
very specific hormone response elements, that is why they bring out transcription activation. So, the identification of glucocorticoid receptors
and disoffering the fact that this hormone go and binds to the receptor and then the
receptor dimerize and they go and bind to specific hormone response element. The cloning of the glucocorticoid receptor
cDNA as well as estrogen DNA are one of the hallmarks or land marks in the area of steroid
hormone receptor signal transaction pathways. So, this marked a very important beginning
in the understanding. How this hormone functions? This actually led foundation for understanding
the functions of many other receptors which we will discuss in the next few classes. Now, what we discussed today is just about
one class of receptor, what are called as type 1 receptors and the estrogen receptors,
glucocorticoid receptors, mineralocorticoid receptor, progesterone and androgen receptor. Now, there is no way huge family which is
called as a nuclear receptor super family and the cloning of these cDNA’s and demonstration
that all this receptors actually have similar domain structure and then people started using
the zinc finger cDNA binding domains of the receptors has proofs and using this proofs,
they started screening cDNA libraries of various tissues. They found that this kind of a domain structure
is present number of other receptors and this led to what is known as a nuclear receptor
super family and is a new nomenclature. This nuclear receptor family includes not
only the steroid hormone receptors; it also includes thyroid hormone receptor, retinoid
acid receptor, what is called as a PPAR and so on and so forth. In the next few classes, we will discuss some
of these receptors and ask some question what is their importance and how do they activate
transcription. So, the characterization of the cloning of
the steroid hormone receptors is one of the very important land marks in the area of the
nuclear receptor biology that actually paved way for the characterization of a number of
other receptors which is mentioned here. So, nuclear receptor super family consists
of not only the steroid hormone receptors marked in red but also number of other receptors. What is very interesting is that many of these
receptors actually we know what kind of ligands bind but there are many other receptors; we
do not know what kind of ligands they actually bind. For example, based on this the nuclear receptor
can actually be classified into three different classes. One is the class 1 receptors which we have
already discussed named steroid hormone receptors which actually bind to palindromic sequences
or inverted repeat sequences and they are usually present in cytoplasm in the hormone
and they are going to the nucleus in the hormone. Tomorrow, in next class we are going to discuss
the class 2 receptors which contain the receptor for a vitamin D thyroid hormones and retinoid
acid and ask the question. How understanding glucocorticoid receptor
function has paved way for the function of this receptors and what kind of sequences
these receptors bind. They are also called what is called as orphan
receptor that is these receptors share the same homology with that of the other receptors. They also contain zinc. They are also zinc finger transcription factors
but we do not know what the ligands for these receptors are. Some of them actually functions of ligand
independent transcription factors and they also bind to the different sequences. So, what I have tried to mention in this class
is to demonstrate the discovery of steroid hormone receptors and how the discovery of
steroid hormone receptor paved way for discovery of the other members of the nuclear super
family. What I have shown in this slide, these three
important people who actually made a wonderful contribution for this steroid hormone receptor
biology. One is Pierre Chambon in France, Ronald Evans
in Salk Institute, California and Elwood Jenson who actually made very pioneering work before
cloning of the receptors actually began and recognizing their important contribution to
this field they were actually awarded called the Albert Lasker basic medical research award. This often refers as the American noble prize
and it is many people working in this era believe that these people actually may deserve
or actually deserve noble prize and may be awarded noble prize in very near in the future
because they made very important contribution for the discovery of the super family of nuclear
hormone receptors and how this nuclear hormone receptors important for a number of physiological
processes. I had a privilege of working in Ron Evan lab
and so I was working in this lab for three years and what we worked is about what is
called cross coupling of nuclear receptors with others in the transcription factors and
we will discuss that later in this course. So, what are the people who made a very important
contribution had a privilege of working with him. I just listed some of the key references which
are listed here which made a very important what are called land mark papers, how receptors
were cloned and how they were identified and if you go through some of this land mark papers,
you will understand and appreciate some of the efforts that has gone into this discovery
of steroid hormone signaling. I think I will stop here.

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