Title

Regulation of Transcription in Animal Cells: A 50-year Journey
Revealing an Expanding Universe of Factors and Mechanisms

Author(s)Robert G. Roeder

Language

EnglishEvent title

The 2021 Kyoto Prize Commemorative Lecture

Publisher

Inamori Foundation

Issue Date

10/01/2022

Start page1

End page

12URL

https://www.kyotoprize.org/wp-content/uploads/2022/10/2021_roeder_en.pdf

URL for Japanese translation: https://www.kyotoprize.org/wp-content/uploads/2022/10/2021_roeder_jp.pdf

The 2021 Kyoto Prize Commemorative Lecture
Robert G. Roeder

Regulation of Transcription in Animal Cells: A 50-year Journey
Revealing an Expanding Universe of Factors and Mechanisms
I am both honored and humbled to receive the 2021 Kyoto Prize in Basic Sciences. It isespecially pleasing in view of the ideals and philosophy of the Inamori Foundation, and I graciously
thank all those associated with the Foundation. I will now describe several major discoveries over
50 years of biological research, as well as some personal background.
It is often stated that we are our genes—but perhaps more accurately, we are the products ofour genes (Fig. 1 ). The central dogma states that DNA is transcribed into RNA copies that arethen translated into proteins with various enzymatic, structural, and regulator y proper ties.
Remarkably, embryonic stem cells can give rise to different cell types with the same set of genes(Fig. 2 ). Related, the normal formation and function of different cell types and many associatedpathologies result from differential gene expression, which is controlled primarily at the level oftranscription, the first step in gene expression. This makes it critical to understand the mechanismsthat regulate transcription, and this has been my major objective, and passion, for over 50 years.
Transcription is carried out by an enzyme called RNA polymerase, and Fig. 3 shows transcription
in prokar yotes as a frame of reference for the eukar yotic transcription that I will describe. In
essence, the DNA is transcribed by a single RNA polymerase, and is regulated by interacting genespecific activators. As is true in eukaryotes as well, transcription begins at a specific site and makes
single-stranded RNA copies of the template strand of the DNA.
As a preview, my major discoveries include eukaryotic RNA polymerases I, II, III, their distinct
str uctures and functions, cognate general initiation factors, the prototype gene-specifictranscriptional activator, general and gene-specific coactivators, causal roles for chromosomal
histone modifications in transcription, and biochemically defined cell-free systems that accuratelytranscribe specific genes. Before discussing these in more detail, a little personal background.

Fig. 1

Fig. 2

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Fig. 3Fig. 4

Fig. 5

Personal Background
I was born and raised on a farm in Southern Indiana. My parents had very limited formal
educations but were caring parents who taught their four children to be honest, humble, anddiligent. And although diligence in school studies was expected, education beyond high school was
not considered, and I was expected to remain on the family farm. Fig. 4 shows a 1946 family photoon a Sunday afternoon outing. Note my older brothers and I, in the lower left, in overalls typicallyworn by farm boys. Fig. 5 shows me learning to drive a tractor in anticipation of future farm work.
As a child, I had little exposure to science per se, but enjoyed tr ying to figure out how small
mechanical devices worked, as well as building various small devices, such as crystal radios. Inhigh school, I was especially interested in mathematics and chemistr y, but had little time forextracurricular activities because of extensive farm chores. Fortunately, with a strong academic
background, I received a full tuition scholarship to attend Wabash College, a small liberal arts
college with strong science departments. Although I had a primary interest in chemistry, I also
became intrigued by biochemistry and the emerging molecular biology during a course taught by
a new assistant professor, Tom Cole, from Caltech. I was especially influenced by the classic 1961
Jacob-Monod paper on gene regulation in bacteria, leading me to think about future studies ongene regulation in animal cells. Those interests led me to a graduate program in biochemistry at
the University of Illinois. At Illinois, I joined the laborator y of Bill Rutter, an inspiring mentor
working on aldolase enzymes and pancreas development, albeit not transcription. Fortunately, heallowed me to initiate studies on transcription, which began after the laborator y moved to the2University of Washington in Seattle in 1965. At that time, virtually nothing was known about gene
regulation in animal cells, except that, as in bacteria, there were three major classes of RNA:messenger, ribosomal, and transfer. My initial studies focused on quantitative measurements of
RNA synthesis in isolated nuclei and in cells during hormonal responses in rat liver and during sea
urchin development. However, it was not yet possible to monitor specific gene products in those
pre-gene cloning days. Therefore, I decided to go to the heart of the transcription problem, and to
first identify the enzyme that transcribes DNA. As detailed later, this led to my discovery of RNApolymerases I, II, III (Pols I, II, III). This was a true Eureka! moment in my career—and also
guaranteed a noteworthy thesis. For postdoctoral studies, I joined the laboratory of Don Brown,
another inspiring mentor, who had purified the large ribosomal RNA genes that I suspected weretranscribed by Pol I because of colocalization in the nucleolus. Surprisingly, I failed to see specifictranscription of these genes by purified Pol I, which led me to suspect that eukaryotic transcription
would be more complicated than imagined and set the stage for subsequent studies in my own
laboratory at Washington University.
Identification of the Eukaryotic Nuclear RNA Polymerases
Returning to my graduate work, and regarding the identification of the eukaryotic nuclear
RNA polymerases, in 1959-1960, Sam Weiss showed NTP-dependent RNA synthesis both inisolated nuclei and in a derived chromatin aggregate. In 1964-1969, several labs reported only asingle chromatographic peak of RNA polymerase activity, suggestive of a single enzyme, but had
employed low salt, low yield extraction procedures. In 1968-1969, in the Rutter laborator y, I
realized that, unlike the situation in bacteria, most RNA polymerase in eukar yotic cells waschromatin-bound, that is engaged in transcription, and systematically developed new extraction
and purification procedures that included: high salt/sonication to dissociate the histone-DNA andpolymerase-DNA-RNA complexes; dialysis to low salt to precipitate DNA-histone, leaving
quantitatively solubilized RNA polymerase; and finally, ion exchange chromatography to resolve
Pols I, II, III. Fig. 6 shows my laboratory notebooks and my thesis with volume 12 containing theoriginal description and characterization of Pols I, II and III. Fig. 7 shows me collecting sea urchins,
the organism in which Pols I, II and III were first discovered. Fig. 8 shows a chromatographic
resolution of the three nuclear RNA polymerases in February of 1969, with the red lines showing
the three different RNA polymerases that were chromatographically eluted. This work resulted in
my first publication, a Nature article (Fig. 9 ). But of note, the paper was submitted to Nature on
August 5, 1969 and initially rejected on the grounds that it was “not of general interest”—not aparticularly happy moment for a graduate student. But happily, it was published on October 18 ofFig. 6

Fig. 7

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that year as originally submitted. The identification of the distinct RNA polymerases in 1969 was
foundational, but as it turns out, and as you will see, just the tip of the iceberg (Fig. 10 )—asdiscussed in my 2003 Lasker Award Commentar y entitled “The eukar yotic transcriptional
machinery: complexities and mechanisms unforeseen”.

Fig. 8Fig. 9Fig. 10

Identification of Specific RNA Polymerase Functions
Having three enzymes, the next task was to identify their specific functions. The earlyfunctional assignments were based on comparisons of the differential sensitivities of the purified
polymerases to the mushroom toxin α-amanitin with the sensitivities of the synthesis of specificRNAs by endogenous, engaged, polymerases in isolated nuclei (Fig. 11 ). As you can see in thepanel on the right, the sensitivity of messenger RNA synthesis matched that of Pol II, thesensitivity of 5S and tRNA synthesis matched that of Pol III, and the insensitivity of ribosomal RNA
synthesis matched that of Pol I. So, these studies led us to the following situation, namely that RNA
polymerases I, II and III transcribe, respectively, the genes encoding ribosomal RNA, messenger
RNA, and 5S and tRNA (Fig. 12 )—and these RNAs converge on the ribosome for protein
synthesis. This scenario is distinct from that in prokaryotes, which have one enzyme for all classesof RNA, and it provides a convenient means for independent regulation of the global synthesis of
the major classes of RNA—for example, during growth state changes.

4Fig. 11

Fig. 12

Assessment of the Structural Basis for the Distinct RNA Polymerase Functions
Given these important findings, the next question was to assess the structural basis for the
distinct RNA polymerase functions. By 1974, my students and I had purified to homogeneity the
RNA polymerases from mouse tumor cells and resolved their subunits by electrophoresis—as
shown in the left panel of Fig. 13. As you can see, the three enzymes have complex and distinct
subunit structures. Twenty years later, the laboratories of Sentenac and Young had purified thehomologous yeast enzymes and cloned the corresponding subunits (Fig. 13, right panel). Thosestudies revealed that some subunits were common to the three RNA polymerases, some werecompletely distinct, and others were highly related to each other and to subunits in the bacterial
RNA polymerase. So, these results revealed a molecular basis for some common enzymatic
properties, as well as the distinct specificities and regulation of the three enzymes.

Fig. 13

Establishment of Accurate Transcription of Specific Genes by Purified RNAPolymerases for Mechanistic Analyses
Given distinct enzyme structures and functions, the next task was to establish accuratetranscription of specific genes by purified RNA polymerases for mechanistic analyses. Thesestudies resulted in the identification of general initiation factors for specific transcription (Fig. 14 ).
Prior to 1979, incubation of purified RNA polymerases I, II, III with corresponding purified genes
failed to yield any specific transcription. But in 1979, we showed that incubation of purified RNA5polymerase II or III with corresponding purified genes in the presence of a cellular extract resulted
in specific transcription. In 1980, the subsequent chromatographic fractionation of this extract
yielded two general initiation factors, TFIIIB and TFIIIC, as well as a gene-specific factor, TFIIIA,
for RNA polymerase III. In the case of RNA polymerase II, the chromatographic fractionation
resulted in the identification of multiple (partially purified) factors that were later resolved into six
independent factors called TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. By 1992, studies in myown and other labs (indicated in Fig. 14 ) had resulted in the complete purification of these factors
and in the cloning and validation of the individual subunits. Fig. 15 shows some of the original data
for accurate transcription initiation by purified Pol II. Incubation of the indicated DNA fragment
containing the adenovirus major late promoter with Pol II and with the extract led to the synthesisof a specific 536 -nucleotide RNA transcript that indicated specific initiation at the natural initiationsite. This RNA was not seen when RNA polymerase II was inhibited with α-amanitin or when theextract was omitted from the reaction. In later studies with purified factors (Fig. 14, lower panel),robust transcription of the same promoter was seen when all factors were present, but not with theomission of any single factor or RNA polymerase II, clearly establishing their requirements forpromoter-specific transcription initiation. An important point from these studies was that our
results showed accurate, but promiscuous, transcription of the cell-specific Adenovirus2 and β-globin promoters by the ubiquitous Pol II and general initiation factors. This led to the prediction,and later discovery, of a general repression mechanism, namely DNA assembly into chromatin,and gene- and cell-specific transcriptional regulatory factors, activators, that reverse the repression.

Fig. 14

Fig. 15

Elucidation of the Mechanism Involved in Specific Transcription Initiation
Having identified general initiation factors, the next task was to establish the mechanism
involved in specific transcription initiation by the polymerase and these factors. Fig. 16 (left panel)summarizes our initial results on a Pol III-transcribed tRNA gene. Promoter recognition isestablished by TFIIIC, and TFIIIC in turn results in the stepwise recruitment of TFIIIB and Pol III,
resulting in the formation of a preinitiation complex containing about 25 polypeptides. Similar
results were obtained for a Pol II-transcribed promoter (Fig. 16, right panel). Our early studies
showed an initial recognition of the promoter by the initiation factor TFIID. Subsequent studies by
my own and other laboratories (indicated in the figure) showed the stepwise assembly of theremaining factors in the formation of a preinitiation complex containing 44 polypeptides. Incubationof either of these preinitiation complexes with nucleoside triphosphates resulted in specifictranscription initiation and subsequent transcription elongation.

6

Fig. 16

Discovery of Gene/cell-specific Transcriptional Activators
The next seminal event in this journey was the discover y of gene- and cell-specifictranscriptional activators. These factors were predicted based on precedent from bacterial studies
and on the promiscuity of the general transcription machinery, necessitating some mechanism toachieve gene- and cell-specific transcription. The first of these factors to be identified was the 5S
gene-specific TFIIIA, which was purified in 1980 on the basis of a functional transcription assay andshown to bind and activate the 5S RNA gene (Fig. 17 ). TFIIIA was the prototype DNA-bindingtranscriptional activator in eukaryotes. The left panel in Fig. 17 shows the transcription assay withpurified Pol III, TFIIIB, and TFIIIC, which result in the robust transcription of the control tRNAgene, but not the 5S RNA gene. Robust transcription of the 5S gene is achieved in the presence of
TFIIIA and these same factors as further shown in the left panel. The center panel simply showsthe electrophoretically resolved, highly purified TFIIIA, and the panel on the right shows promoter
binding of TFIIIA by a DNase footprint assay. These studies in 1980 were followed by oursubsequent cDNA cloning of TFIIIA, providing the first protein sequence of a transcription
initiation factor and leading Aaron Klug to deduce the zinc finger motif, which is the most common
DNA binding motif in eukaryotic transcription factors. Mechanistically, we showed that TFIIIA
binding to the promoter facilitates the subsequent recruitment of TFIIIC, which otherwise does
not bind to this particular promoter, and that TFIIIC, in turn, facilitates the stepwise recruitment of
TFIIIB and Pol III, as described for the tRNA gene (Fig. 18 ). This represents the first definedmechanism of action for any gene-specific transcriptional activator in eukaryotes, and is distinct
from the prokaryotic mechanism in which activators directly bind the RNA polymerase (Fig. 3 ).

Fig. 17

Fig. 18
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Subsequent studies in my own and other laboratories in the next four years led to theidentification of four gene-specific activators for Pol II-transcribed genes (Fig. 19 ), and currently
we know there are about 1,600 of these gene-specific, cell-specific factors. They are typified by a
DNA binding domain and a so-called activation domain. Many of these factors are mastertranscriptional regulators of cell fate and differentiation, as summarized in Fig. 20. Weintraub, in
1987, showed that MyoD could convert a fibroblast to a muscle cell. Yamanaka, in 2006, showedthat a fibroblast could be converted to a pluripotent stem cell by the ectopic expression of only fourother factors. These remarkable studies were recognized by the Kyoto Prize, the Nobel Prize, andothers. These studies and other studies with master transcriptional regulators, some of which are
shown in Fig. 20, emphasize both the physiological significance and the power of thesetranscription factors—their ability to change cell fate.

Fig. 19

Fig. 20

Establishment of the Mechanism of Action of Gene/cell-specific Activators
Given their extreme biological significance, the next task was to establish the mechanism ofaction of gene- and cell-specific activators for Pol II-transcribed genes. The basic question here wasonce an activator is bound to the promoter or enhancer site, how does it effect the formation andfunction of the preinitiation complex on the target gene (Fig. 21 )? Quite surprisingly, given the
structural complexity of these entities, the activators failed to function with highly purified Pol II
and initiation factors. Functional biochemical assays then identified essential coactivators in theearly 1990s in several laboratories, some of which are summarized in Fig. 22. This introduces agroup of cofactors that operate directly on the general transcription machinery. These cofactors
include the TAF subunits of TFIID, which were initially described in Drosophila by the Tjian lab
and in human cells by the Roeder and Berk labs. The 30-subunit Mediator complex, probably themost impor tant coactivator complex, was initially described in yeast based on genetic andbiochemical assays by the Young and Kornberg labs, and shown to interact directly with RNA
polymerase II. It was first described in human cells by our laboratory on the basis of biochemical
assays, which also showed a direct binding to transcriptional activators. Both of these groups ofcoactivators, the TAFs and the Mediator, are generally required for activator function. We also
identified OCA-B, a B cell-specific coactivator that is selective for the OCT 1 - and OCT 2 -boundgenes in B cells and the first representative of an expanding class of cell- and gene-specificcoactivators. Fig. 23 shows more detail on the Mediator mechanism, which basically acts as abridge between diverse enhancer-bound activators and the basal transcription machinery. As we
initially showed, liganded nuclear hormone receptors like TR and PPARγ interact with theMediator through the MED 1 subunit, according to the detailed interactions shown in the figure.

8This direct interaction serves to recruit the Mediator to activator-bound enhancers for subsequentinteractions with the general transcription machinery at the promoters. Again, this model was
based on biochemical assays. It was validated by a MED 1 knockout in a mouse embryo fibroblastbased model of PPARγ-dependent adipogenesis, as shown in Fig. 24. As shown originally in theSpiegelman lab, fibroblasts can be differentiated to adipocytes, fat cells, with inducing factors andthe master regulator PPARγ; and those cells, differentiated adipocytes with lipid droplets stainedby Oil Red O, are shown in the left panel. As shown in the right panel, the MED 1-deficient MEFs
fail to differentiate into adipocytes under these same conditions, and they further show impaired
PPARγ target gene expression. So, this analysis provided a validation of the function andphysiological relevance of the Mediator, and specifically the MED 1 subunit.

Fig. 21

Fig. 22

Fig. 23

Fig. 24

Transcriptional Regulation in a Chromatin Context
Given its natural intracellular locale, we next analyzed transcriptional regulation in the context
of chromatin. As is well known, genomic DNA interacts with core histones to form nucleosomal
structures that both package DNA and, as you will see, serve to repress transcription (Fig. 25 ).
Remarkably, many laboratories showed that the histone tails, on a nucleosome, can be acetylated,
methylated, phosphorylated, and ubiquitylated at very specific sites by a diverse group of enzymes.
This brings us to a group of cofactors that act through modifications of the chromatin template(Fig. 26 ). These cofactors include ATP-dependent chromatin remodeling factors and histone
modifying factors that generate the indicated modifications. These factors were discovered by9other laboratories, but we were basically interested in establishing a defined cell-free system toinvestigate the integrated functions of these cofactors for both biochemical and mechanistic
studies. The general strategy is shown in Fig. 27. As already described, incubation of a DNA
template with Pol II and initiation factors results in robust—albeit promiscuous—accuratetranscription. My laboratory and the laboratories of Kornberg and Luse showed that the assemblyof this DNA into a chromatin structure with histones resulted in the repression of this promiscuoustranscription activity. So, basically, this established the general repression mechanism that had
been predicted. These templates and our assays were then utilized in conjunction with Pol II,
initiation factors, various activators, and various cofactors to establish mechanisms involved in thereactivation of the native, repressed chromatin template. More detail for this protocol is shown in
Fig. 28. A DNA template with activator sites and a core promoter is assembled into chromatin withbacterially expressed and purified histones according to the procedure of Jim Kadonaga. This
chromatin, which resembles a “beads-on-a-string” structure, is then incubated with activators andwith acetyl and methyl transferases to effect chromatin modifications. Those modified templates
are then incubated either with nuclear extract as a source of all of the other general transcription
factors or, alternatively, with purified Pol II, initiation factors, coactivators, and elongation factors toprovide a quite defined in vitro transcription system, with a chromatin template, that contains inexcess of a hundred different polypeptides. Notably, the chromatin can be assembled with wildtype histones, with mutant histones that cannot be modified, or with pre-modified histones. Thissystem has allowed us to establish causal effects of histone modifications as well as direct effects
and mechanisms of action of various coactivators. Some of these results are summarized in Fig. 29.

Fig. 25

Fig. 26

Fig. 27

Fig. 2810

In early studies, we analyzed the function of p300, a histone H 3/H 4 acetyltransferase, in
conjunction with two different arginine methyltransferases that effect specific modifications. We
later analyzed the function of p 300 in conjunction with the SET 1 complex, a major lysine histone
methyltransferase that modifies lysine 4 in the promoter regions. In general summary of thesestudies (Fig. 29 ), they showed ordered, cooperative interactions and functions of coactivators inactivator-dependent transcription along with specific histone modifications. Perhaps mostimportantly, they established causal effects of histone modifications on transcription. This wasindicated by the observation that mutations in the modified histone acetylation and methylationsites eliminated the cofactor functions. This observation was critical, since we, in 1997, and later
others, showed that histone modifying factors can also functionally modify many transcription
factors. And the more common cell-based and genetic assays show only correlations of histone
modifications with transcription and do not identify the essential substrates. So, again, the studies
were important for establishing the anticipated causal effects of histone modifications.

Fig. 29

Principal Discoveries and Achievements
Fig. 30 provides a general summar y of these discoveries, which include: structurally andfunctionally distinct RNA polymerases, cognate general initiation factors, gene- and cell-specifictranscriptional activators, general and gene- or cell-specific cofactors, mechanisms involved in theregulation of transcription through the use of biochemically defined systems with purified factors
and recombinant DNA and chromatin templates, and a chromatin-based repression mechanism aswell as causal roles for histone modifications in transcription. The diagram in Fig. 30 gives you avisual perception of the overall complexity of the factors and complexes required for thetranscription of a simple single gene, requiring at least a hundred or more distinct polypeptides
dispersed among the various factors.
These discoveries have been foundational for subsequent and future studies of the spectacularhigh resolution X-ray/cryo-EM studies of the transcriptional machineries, genomic analyses ofgene activation mechanisms, cellular imaging studies of gene activation mechanisms, mechanisms
underlying distal enhancer-promoter interactions and functions, an emerging role of phaseseparation (biological condensates) in gene activation, transcriptional regulator y circuits, and,
importantly, the molecular basis and the therapeutic manipulation of aberrant transcription factor
functions and transcriptional regulatory circuits found in many human pathologies.

11Fig. 30

Acknowledgements
In closing, I would like to acknowledge over 120 graduate and postdoctoral trainees who havecontributed to this work, my undergraduate, graduate and postdoctoral mentors, my manycolleagues in the transcription field, and my family—who have unfailingly supported my dedication
to science. Fig. 31 shows three of my inspiring mentors in my early career development: my
Wabash College professor Tom Cole, my Ph.D. mentor Bill Rutter, and my postdoctoral mentor
Donald Brown. Fig. 32 shows some of my extraordinary students and postdoctoral trainees on theoccasion of my 70 th birthday celebratory symposium in 2012 at The Rockefeller University. And
lastly, I would again like to acknowledge the Inamori Foundation for this truly remarkable award.

Fig. 31

Fig. 3212