C8545 Developmental Biology
Lesson 10
Regulation of Gene Expression during Development
Jan Hejátko
Functional Genomics and Proteomics of Plants
CEITEC
and
National Centre for the Biomolecular Research,
Faculty of Science
Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.eu
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Literature
 Fred H. Wilt and Sarah Hake, Principles of Developmental Biology
(W.W. Norton & Company, New York, London, 2004)
 Capron A, Chatfield S, Provart N, Berleth T 2009. Embryogenesis:
Pattern Formation from a Single Cell. The Arabidopsis Book. Rockville,
MD: American Society of Plant Biologists, doi: 10.1199/tab.0126,
http://www.aspb.org/publications/arabidopsis/.
 Selected original papers in scientific journals
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
 Protein localization
 RNA interference
 Identification and mechanism of gene expression regulation
via RNA interference
 siRNA-mediated silencing
 miRNA-mediated silencing
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
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The regulation of gene expression during development may occur at different levels.
Most of the regulations occurs at the transcriptional levels, however, the later
(“downstream”) type of posttranscriptional regulations e.g. regulation of translation,
posttranslational modifications of the proteins, their transport etc. are also important.
Recently, very important type of posttranscriptional regulation was found to be
mediated by small RNAs. The molecular mechanisms and their importance for the
diverse developmental processes in eukaryotes with emphasis on the development
regulation in plants will be discussed in the second part of this lesson.
The regulatory events are diagrammed here as a linear sequence, which, however,
is rarely the case. More often, the individual regulatory events occur in a maze of
networking interactions.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
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Regulation by
histone acetyl
transferases or
histone
deacteylases
Regulation of the chromatin structure represents one of the very basal gene
expression regulatory levels. Chromatin is a substrate for DNA-dependent RNA
polymerases that transcript the DNA encoded information into the “words and
sentences” of RNA.
Regulation of chromatin structure and its accessibility to DNA-dependent RNA
polymerases depends on many factors, one of the most important is the regulation
of chromatin binding to nucleosomes and chromatin methylation.
Regulation of chromatin interaction with histones, the positively charged proteins
forming the core of nucleosomes, is performed via modification of acetylation status
of the N-terminal portion of histones, especially histones H3 and H4. This occurs via
action of histone acetyl transferases or histone deacteylases.
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CpG or CpNpG
CpNpNp
CpG
DNA methylation in animals vs. in plants
methylation status methylation status
Cell-specific methylation allows maintain of tissue-specific gene
expression profiles
Mechanism of transcriptional regulation by
DNA methylation mostly unknown
Modification of the chromatin methylation is performed via DNA methyltransferases.
Interestingly, there is difference in the methylation in animals and in plants.
In animals, the methylation occurs mostly on the cytosine that occurs next to
guanosine (the sequence is denoted as CpG). In mammals, 60-90% of all CpGs are
methylated.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and
asymmetrically (CpNpNp), where N is any nucleotide.
Methylation status is usually “reset” in the zygote and is reconstituted during
development again. E.g. the methylation is very low in the mouse embryo at the
blastula stage, however, DNA derived from later stages when organogenesis is
initiated is substantially more modified by methylation.
DNA methylation also stably alters the gene expression pattern in cells such that
cells can "remember where they have been"; in other words, cells programmed to
be pancreatic islets during embryonic development remain pancreatic islets
throughout the life of the organism without continuing signals telling them that they
need to remain islets.
DNA methylation is involved in the genomic imprinting, i.e. the genes originating
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from both parents are often diversely methylated, which results into differential
expression of parental genomes (for the importance of the imprinting in the parental
conflict and epigenetics, see the lecture “Bi0580 Developmental genetics” by prof.
Vyskot).
Up to know it is not clear how methylation regulates transcription. Possibly,
methylation status affects chromatin configuration or binding general repressor
factors.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
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Formation of transcription initiation complex
Regulation of transcription occurs via specific interaction of both general and tissue
specific transcription factors (TFs) with promoter and/or enhancer sequences.
The scheme above shows simplified subsequent formation of the complex of TFs
involved in the regulation of transcription. Interaction of general TFIID with the TATA
box induces distortion of the DNA structure (see the next slide).
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Induction of structural changes upon interaction of TFIID with DNA. This may be
important for the assembly of other TFs involved in the formation of transcription
initiation complex.
This change of confirmation provides a kind of “signature” that is recognized by
other proteins and NA polymerase to recognize the proper binding site. However,
there are also TATA box-less promoter, where probably other types of “signatures”
occur.
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Positive TFs
Negative TFs
Formation of transcription initiation complex
The scheme showing the formation of the transcription initiation complex and the
interaction of both positive (open symbols) and negative (solid symbols) factors.
These proteins bind to the regulatory sequences that might be hundreds or even
thousands of base pairs away from the promoter. These protein interact with each
other and with the RNA polymerase, integrating thus many signals into a “yes” or
“no” response of the basal promoter, i.e. the region adjacent to the TATA box and
recognized by the RNA polymerase.
The individual positive or negative factors are complex and their activity might be
regulated by their phosphorylation status or via their interaction with other proteins
(i.e. monomeric or dimeric) etc..
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Mechanism of transcriptional regulation
by TAFs
Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
There is a whole family of transcription activating factors (TAFs) that interact with
signalling molecules, e.g. steroid hormones, thyroid hormones or retinoic acid and in
a response to the signal translocate to the nucleus and activate transcription.
One of the type of TAF are leucine zipper or bZIP type TAFs. These TAFs are
dimeric, with leucine-rich hydrophobic face formed by the Leu that occurs every 7th
aa.
That allows the factor to take the proper configuration, which provides the dimer
with the ability to bind DNA via charged aa.
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“Microprocessor-like” acting promoters
ProENDO16:REPORTER
Deletion
mutagenesis Positive, interaction with
TAFs
Upregulation in the
presence of A and B
Developmental specificity
Combinatorial control
An example of the “microprocessor”-like acting promoter is a promoter of the endo16 gene from the
sea urchin.
Endo16 is a single copy gene that codes for the RGD-containing calcium-binding protein of the cells
of invaginating archenteron during gastrulation.
As visualized in experiments, the Endo16 protein may be an adhesion molecule, involved in the
gastrulation of the embryo (Nocente-McGrath C et al, 1989).
There have been identified several gene regulatory modules in the endo16 gene that have positive or
negative regulatory role. These modules were identified via formation of deletion mutants of the
transcriptional fusions with reporter gene.
The analysis has revealed that the module A has a positive function and must interact with its
cognate TAFs for transcription to occur.
Module G enhances the expression when the A and B are active.
C, D, E and F are responsible for the specificity of the expression of endo16 during sea urchin
development.
Each of the modules has several protein interaction sites, some of them general, other unique. Site
for the protein SpGCF1 is present in many modules and is probably responsible for looping of
chromatin, allowing thus bringing of distal regulatory modules close to the basal promoter.
This type of regulation, i.e. based on the different activities of diverse regulatory sequences is
sometimes called combinatorial and is common for development of many living creatures.
In the combinatorial type of regulations, some modules may act synergistically, some of them
antagonistically, some may have both positive and negative roles (e.g. the module B, see the figure).
This variability allows very precise and responsive regulations towards changing environmental
conditions.
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“Microprocessor-like” acting promoters
Regulation of β-globin type of
hemoglobin chains expression
Locus control region
Development-dependent
activation by LCR
•Acetylation of H3?
•Involvement of other
genes?
Cca 50 kbp
An example of the combinatorial gene regulation is the regulation of β-globin type of hemoglobin
chains of humans.
As discussed in the Lesson 5, the type of hemoglobin produced by the fetus changes during
development. The hemoglobin present in the liver-produced hemoglobin is composed of two α- and
two β-type chains. The β-type hemoglobin chains are of several developmental types, produced by ε,
γ1, γ2 and β (in this order). In addition, there is minor adult type of β-type hemoglobin, called δ
globin.
The genes for the β-type chains are aligned on the chromosome in the order, in which they are
expressed during development (se the figure).
For the expression of individual cell types is distinctive an upstream regulatory sequence called locus
control region (LCR). LCR is located about 50 kbp away from the most proximal ε gene.
The LCR structure is different in erytrocyte precursor cells in comparison to other cells that could be
demonstrated by the changes in the sensitivity to low concentrations of DNase, suggesting low
amount of nucleosomes bound.
For the expression of the particular genes, the interaction of their regulatory sequences with LCRs is
necessary. Because of LCR can interact only with one regulatory sequence at a time, only one type
of genes for the particular β-type chain is activated (the first interaction of LCR with ε gene, which is
later in development replaced by the other one, is shown by the double-headed arrow).
The underlying molecular mechanisms of the specific pattern of the LCR movement from the most
proximal towards the most distal gene cannot be satisfactory explained.
Probably, acetylation of H3 histones might play a role and possibly, other genes outside of the β-type
chain family are involved in the regulation of LCR activity. That seems to be confirmed by the
identification of other human genes with similar structure, suggesting common regulatory
mechanisms via LCRs.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
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Splicing of hnRNA
Posttranscriptional modifications takes place after transcription and these are a
matter of regulation that has developmental consequences, too.
The most important posttranscriptional modification in eukaryotes in splicing of
introns. Immediately aftre transcription, the hnRNA is polyadenylated and interacts
with proteins and small nuclear RNAs (snRNAs) that are part of small nuclear
nucleoproteins. These interact with specific recognition sites and allow splicing of
introns and connection of exons into final mRNA.
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Sex-specific splicing of DOUBLE SEX (DSX) hnRNA in Drosophila
hnRNA
Inhibition of male-type differentiation
Inhibition of female-type differentiation
An example of the developmental importance is an alternative, sex-specific splicing
of DOUBLE SEX (DSX) gene in Drosophila. DSX is a last member of the cascade
involved in the sex specification of Drosophila.
In females, exons 1 through 4 are connected, while in males, exons 1-3, 5 and 6 are
connected into final mRNA that is translated.
Male form of DSX blocks the female-type of differentiation and vice versa, the
female form of DSX blocks the male-type of differentiation.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
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Translation initiation after egg fertilization
Initiation of translation, elongation of the peptide chain and protein release are all
the steps being regulated during development.
An example is translation initiation after fertilization e.g. in the sea urchin eggs. On
the figure above, there is shown a dramatic increase of polyribosomes formation
(polyribosome is a string of ribosomes on mRNA) and increase in the protein
synthesis after urchin egg fertilization.
The polyribosomes form on the preexisting mRNA, originating from the egg.
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Developmental Biology 8e Online (http://8e.devbio.com/)Puoti et al., EMBO Rep (2001)
Inhibition of translation initiation via
mRNA masking
Cessation of
sperm cells
production
TRA2
3’ untranslated region (3’ UTR) of mRNA could interact with specific proteins. This
interaction then leads to the inhibition of translation initiation and the process is
called masking of mRNAs.
This mechanism occurs in many organisms, including Xenopus, sea urchin, mouse
and Coenorhabditis.
The masking regulates also polyA tail length. Once unmasked, the mRNA could be
intensely polyadenylated, leading to increase of the polyA tail.
The length of the polyA tail also affects translation initiation and stability of mRNA.
On the figure above, there is shown a mechanism of the sex determination in
Coenorhabditis, where posttranslational regulation of expression plays important
role.
Most C. elegans have a female body but are hermaphroditic, producing both sperm
and eggs at different times.
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The first germ cells to differentiate in the nematode become sperm, which are stored
in the uterus for later use. After the fourth molt (from larva to adult), the germ cells
cease making sperm and begin to make eggs. These eggs will eventually become
fertilized by the stored sperm.
The process determining which path the germ cell follows—to sperm or to egg—
depends on the translational repression of different messages.
The initiation of sperm formation is achieved by the repression of the tra-2 message.
The Tra-2 protein is essential for the development of eggs and female body cells, and
repression of tra-2 mRNA translation in germ cells causes them to become sperm.
The 3' UTR of this message contains two regions of 28 nucleotides, each of which
appears to bind a putative repressor protein that is synthesized during the larval
stages associated with spermatogenesis. If these regions are mutated, the translation
of the tra-2 mRNA is not repressed, no sperm is made, and the nematode is
functionally female instead of hermaphroditic .
The switchover from spermatogenesis to oogenesis also requires suppressing the
translation of the fem-3 gene through its 3' UTR.
The Fem-3 protein is critical for specifying male body cells and sperm production.
Transcription of the fem-3 gene is inhibited by Tra-2 protein, but the repression of
existing fem-3 messages is also needed. This translational repression appears to be
effected by the binding of a translational inhibitor by the 3' UTR of the fem-3 mRNA.
Thus, the initiation of spermatogenesis in hermaphroditic nematodes and the
transition from spermatogenesis to oogenesis appears to be regulated by
translational repression through the 3' UTR.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
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Regulation via mRNA localization
Binding of
STAUFEN
Dimer structure of two BICOID
mRNAs recognized by STAUFEN Developmental Biology 8e Online (http://8e.devbio.com/)
Messenger mRNA may be localized to the specific positions in the cell. Specific
mRNA localization was discussed in the Lesson 2, when during syncitial stage of
the Drosophila embryo formation, BICOID mRNA is located to the dorsoanterior
portion of the developing embryo.
Again, as just discussed, 3’ UTR plays an important role in the tethering of BICOID
mRNA.
Transcription of the bicoid message occurs in the nurse cells (during stages 4 and 5
of oogenesis) and the mRNA is transported immediately into the oocyte.
BICOID mRNA is localized to the anterior end of the oocyte with the aid of
STAUFEN protein. STAUFEN recognizes regions of double stranded mRNA, which
are present in arm III of the BICOID 3' UTR (see left-hand figure). The STAUFENBICOID
mRNA complex associates with the minus ends of the microtubules,
thereby localizing BICOID to the anterior.
On the right-hand figure, there is a secondary structure of the BICOID mRNA
3'UTR.
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STAUFEN protein binds to specific sequences of arms III, IVb and Vb shown here in
blue lettering.
Helix III is essential for the binding to STAUFEN protein and accumulation in the
anterior of the oocyte. The insert shows the interactions between helices III of two
BICOID mRNAs. This structure is recognized by the STAUFEN protein.
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Regulation via mRNA localization
Another example represents localization of NANOS mRNA (see the figure above).
NANOS mRNA is localized posteriorly and acts as a translational suppressor of the
HUNCHBACK protein (recall the Lesson 2 and next slides).
HUNCHBACK is a TF that further acts as a regulator of other donwnstream genes,
e.g. KRUPPEL (KR), KNIRPS (KNI) and GIANT (GT) (see next slides).
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The levels of BCD and NANOS at the time of fertilization.
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NANOS+PUMILO
HUNCHBACK
mRNA
deacetylation and
degradation
After fertlization, the proteins are translated. NANOS inhibits HUNCHBACK translation
posteriorly.
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Later in development, HUNCHBACK is induced anteriorly by BCD.
The gradient of HUNCHBACK regulates the position of expression of KRUPPEL (KR),
KNIRPS (KNI) and GIANT (GT).
HUNCHBACK upregulates KR and KNI, where KR requires higher than maternal level of
HUNCHBACK .
GT is repressed by HUNCHBACK.
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Regulation via mRNA localization
NANOS+PUMILO
NANOS mRNA requires for its function in a translational repression of
HUNCHBACK another protein, PUMILO.
PUMILO binds to the 3’UTR of HUNCHBACK mRNA and allows thus binding of
NANOS and subsequent deadenylation of the HUNCHBACK and its degradation.
Localization of NANOS mRNA is under control of several genes (e.g. OSKAR,
STAUFEN, VALOIS, VASA and TUDOR). The proteins of these genes bind top the
3’UTR of NANOS mRNA and allow its posterior localization.
Interestingly, the localization of NANOS mRNA is predominantly at the posterior
pole, but not exclusively. There is still some NANOS located in the middle or even
anterior portion of the embryo. However, only posteriorly located NANOS is
translated.
That is ensured by the action of protein SMAUG. SMAUG binds to the specific
sequence in the 3’UTR of NANOS mRNA and inhibits its translation. The localization
of mRNA to the posterior domain relieves this translational repression (see the
figure).
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
 Protein localization
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Leaving the Golgi apparatus
Esterification and cholesterol addition
Regulation via protein localization
Autocatalytic cleavage and esterification represent another example of
posttrransaltional modifications during development.
HEDGEHOG (HD) protein from Drosophila is important signalling ligand involved in
the formation of boundaries from body segments and other patterned elements
during development.
HD contains the signal peptide that directs the HD protein to the ER and later to the
Golgi apparatus. After leaving the Golgi apparatus, the protein is cleaved into two
parts: The N-terminal (19 kD) and C-terminal portion (25 kD).
The N-terminal portion is later modified via esterification and cholesterol is added to
its C-terminus. Only the N-terminal part acts as an active protein that bids to its
receptor.
Modification of HD via cholesterol binding has an important role in the regulation of
the HD diffusion from the HD expressing cells. Genes like DISPATCHED or TOUT
VELU do affect mobility of HD that is important for HD-mediated signalling.
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Regulation via protein localization
Similar function of cholesterol as in case of HD protein seems to be applied also in
the case of its vertebrate homologue, SONIC HEDGEHOG (SHH), discussed
previously (see Lesson 4) during the formation of dorsolateral axis in the neural tube
development.
Notochord acts as a source of the SHH production that leads to the differentiation of
floor plate in the ventral portion of the neural tube while motor neurons differentiate
in the dorsolateral poprtion, where SHH concentration is much lower.
Probably, the cholesterol modification is involved in the regulation of SHH
distribution.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
 Protein localization
 RNA interference
 Identification and mechanism of gene expression regulation
via RNA interference
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RNAi rnai
Mello and Conte, Nature (2004)
RNA interference as a natural mechanism of the gene expression
RNA interference or RNAi, is a mechanism of targeted posttranscriptional regulation of
gene expression, what is also called posttranscriptional gene silencing (PTGS).
The molecular mechanism o PTGS was discovered in experiments with gene silencing in
Coenorhabditis elegans. It was found that surprisingly both sense and anti-sense mRNA
when injected into Coenorhabditis was able to silence the gene of interest.
One of possible hypothesis predicted that contamination during in vitro transcription might
lead to the formation of dsRNa that would be responsible for the observed phenomenon.
The hypothesis was confirmed by the introducing dsRNA in Coenorhbditis and it was found
that the dsRNa induced gene silencing by about an order or two better then anti sense
mRNA.
Importantly, in then forward genetic screens were identified mutants that were affected in
the dsRNA-induced gen silencing. This suggested that the mechanism of dsRNA-mediated
gene silencing is an intrinsic regulatory mechanism for regulation of gene expression.
That finding triggered an explosion of subsequent discoveries and led to the identification of
many members of the entire pathway.
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Mello, 2004
RNA-dependent RNA polymerase
short hairpin RNA micro RNA
Mello and Conte, Nature (2004)
Mechanism of RNA interference
+ tasiRNAs
21-24 bp
It has been found that dsRNA might be either an intermediate or a trigger in PTGS.
In the first case, dsRNA is formed by the action of RNA-dependent RNA polymerases (RdRPs), which use specific transcripts as a template. It
is still not clear, how these transcripts are recognized, but it might be e.g. abundant RNA that is a result of viral amplification or transcription of
foreign DNA.
It not clear, how the foreign DNA might be recognized, possibly, lack of bound proteins on the foreign “naked” DNA and its subsequent
“signature” (e.g. by specific methylation pattern) during packing of the foreign DNA into the chromatin structure might be involved.
The highly abundant transcripts might be recruited to the RdRPs by the defects in the RNA processing, e.g. lack of polyadenylation.
In the case when dsRNA is a direct trigger, there are two major RNA molecules involved in the process: Short interference RNA (siRNA) and
micro RNA (miRNA), both encoded by the endogenous DNA.
These two functionally similar molecules differ in their origin:
siRNAs are dominantly product of the cleavage of the long dsRNA that are produced by the action of cellular or viral RdRPs. However, there
are also endogenous genes, e.g. short hairpin RNAs (shRNAs) allowing production of the siRNA (see the figure).
miRNAs are involved in the developmental-specific regulations and are product of transcription of endogenous genes encoding for small
dsRNAs with specific structure (see the figure).
In addition to siRNAs, there are trans-acting siRNAs (tasiRNAs) that are a special class of siRNAs that appear to function in development (much
like miRNAs) but have a unique mode of origin involving components of both miRNA and siRNA pathways.
Developmental regulations via miRNAs are more often used in animals then in plants.
The dsRNAs of all origins and pre miRNAs are cleaved by DICER or DICER-like (DCL) enzyme complexes with RNAse activity, leading to
production of siRNAs and miRNA, respectively.
These small RNAs are of 21-24 bp long and bind either to RNA-induced transcriptional silencing complex (RITS) or RNA-induced silencing
komplex (RISC).
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From MacRae, I.J., Zhou, K., Li, F., Repic, A., Brooks, A.N., Cande, W.., Adams, P.D., and Doudna, J.A. (2006) Structural basis
for double-stranded RNA processing by Dicer. Science 311: 195 -198. Reprinted with permission from AAAS. Photo credit: Heidi
Dicer and Dicer-like proteins
In siRNA and miRNA biogenesis, DICER or DICER-like (DCL) proteins cleave long
dsRNA or foldback (hairpin) RNA into ~ 21 – 25 nt fragments.
Dicer’s structure allows it to measure the RNA it is cleaving. Like a cook who “dices”
a carrot, DICER chops RNA into uniformly-sized pieces.
Note the two strands of the RNA molecule. The cleavage sites are indicated by
yellow arrows.
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Reprinted by permission from Macmillan Publishers Ltd: EMBO J. Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and Benning, C. (1998) AGO1
defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17: 170–180. Copyright 1998; Reprinted from Song, J.-J., Smith, S.K., Hannon, G.J., and
Joshua-Tor, L. (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305: 1434 – 1437. with permission of AAAS.
Argonauta argo
Argonaut pelagickýago1
Argonaute proteins
ARGONAUTE proteins bind small RNAs and their targets.
ARGONAUTE proteins are named after the argonaute1 mutant of Arabidopsis; ago1
has thin radial leaves and was named for the octopus Argonauta which it resembles
(see the figure).
ARGONAUTE proteins were originally described as being important for plant
development and for germline stem-cell division in Drosophila melanogaster.
ARGONAUTE proteins are classified into three paralogous groups: Argonaute-like
proteins, which are similar to Arabidopsis thaliana AGO1; Piwi-like proteins, which
are closely related to D. melanogaster PIWI (P-element induced wimpy testis); and
the recently identified Caenorhabditis elegans-specific group 3 Argonautes.
Members of a new family of proteins that are involved in RNA silencing mediated by
Argonaute-like and Piwi-like proteins are present in bacteria, archaea and
eukaryotes, which implies that both groups of proteins have an ancient origin.
The number of Argonaute genes that are present in different species varies. There
are 8 Argonaute genes in humans (4 Argonaute-like and 4 Piwi-like), 5 in the D.
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melanogaster genome (2 Argonaute-like and 3 Piwi-like), 10 Argonaute-like in A.
thaliana, only 1 Argonaute-like in Schizosaccharomyces pombe and at least 26
Argonaute genes in C. elegans (5 Argonaute-like, 3 Piwi-like and 18 group 3
Argonautes).
http://youdpreferanargonaute.com/2009/06/
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MIR gene
RNA Pol
AAAn
AGO AAAn
RNA Pol
mRNA
AGO
AGO
RNA Pol
AGO
AGO
AAAn
siRNA
miRNA
post-transcriptional gene silencingtranscriptional gene silencing
transcriptional slicing
translational repression
binding to DNA
binding to specific
transcripts
MicroRNAs are encoded by MIR genes, fold into hairpin structures that are
recognized and cleaved by DCL (Dicer-like) proteins.
In summary, siRNAs-mediates silencing via post-transcriptional and transcriptional
gene silencing, while miRNAs -mediate slicing of mRNA and translational
repression.
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The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire Craig C. Mello
USA USA
Stanford University
School of Medicine
Stanford, CA, USA
University of
Massachusetts Medical
School
Worcester, MA, USA
b. 1959 b. 1960
In 2006, Andrwe Z. Fire and Craig C. Mello were honored by the Nobel prize “for
their discovery of RNA interference - gene silencing by double-stranded RNA“.
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Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
 Protein localization
 RNA interference
 Identification and mechanism of gene expression regulation
via RNA interference
 siRNA-mediated silencing
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Transcription
DNA
Histone proteins
Silencing via covalent DNA
modifications or histones
Transcriptional gene silencing via covalent modifications of DNA
Frequently associated with stable
heterochromatin (centromeres)
Small RNAs can initiate gene silencing through covalent modifications of the DNA or
its associated histone proteins, interfering with transcription.
This form of silencing is frequently associated with stably silenced DNA including
centromeres and transposons, but also occurs at genes.
This figure represents chromatin (DNA wrapped around histones) in an open and
closed conformation. Chromatin modifications include covalent modifications to DNA
and the histone proteins.
40
41
O N
NH2
N
~
O
N
N
NH2
~
CH3
cytosine 5-methylcytosine
DNA methylation
DNA methyltransferase
Histone modification
• molecular mechanism unknown
• involvement of RNA polymerase IV a V
Transcriptional gene silencing via DNA methylation
siRNAs can target DNA for silencing by cytosine methylation or histone modifying
enzymes.
DNA can be covalently modified by cytosine methylation, carried out by DNA
methyltransferases.
The precise mechanisms by which siRNAs target DNA for silencing are not known,
but involve the action of two plant-specific RNA-polymerase complexes, RNA
Polymerase IV (Pol IV) and RNA Polymerase V (Pol V).
41
42
Complex Distribution Function
RNA Polymerase I All eukaryotes Production of rRNA
RNA Polymerase II All eukaryotes Production of mRNA,
microRNA
RNA Polymerase III All eukaryotes Production of tRNA, 5S
rRNA
RNA Polymerase IV Land plants Production of siRNA
RNA Polymerase V Angiosperms Recruitment of AGO to
DNA
DNA
RNA
RNA
Polymerase
Plants have additional RNA Polymerase complexes that contribute to silencing.
Besides RNA polymerases I-III that occur also in other eucaryotes, plant posses
RNA polymerase IV that is involved in the siRNAs and RNA polymerase V that is
necessary for the recruitment of AGO to DNA (see the table above).
42
43
From Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005) RNA polymerase IV directs
silencing of endogenous DNA. Science 308: 118–120. Reprinted with permission from AAAS.
Arabidopsis with silenced
GFP gene
nrpd1a-1
Loss of function of an RNA Pol IV gene interferes with
silencing
In the wild-type background, the red indicates endogenous chlorophyll fluorescence
with no GFP expression; it is silenced.
NRPD1A encodes a subunit of RNA Polymerase IV. In the nrpd1a mutant, RNA
Polymerase IV isn’t produced, interfering thus with silencing.
Green signal (arrow) indicates GFP is expressed, showing that Pol IV is required for
gene silencing.
43
44
DNA
methylation
Histone
modification
DICER-
mediated
siRNA
production
Binding of
siRNA to AGO
Targeting of
silencing
machinery to
the target via
non-coding
transcripts
RNA Pol IV and V are necessary for transcriptional
silencing
RNA Pol IV contributes to siRNA production. Non-coding RNAs produced by RNA
Pol V direct silencing machinery to target sites.
44
45
Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., Givan, S.A., and Carrington,
J.C. (2007) Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57.
Abundance of
small RNAs
Abundance of
transposon/
retrotransposons
Chromosome
Centromere
Most siRNAs are produced from transposons and repetitive
DNA
Most of the cellular siRNAs are derived from transposons and other repetitive
sequences. In Arabidopsis, as shown above, there is a high density of these repeats
in the pericentromeric regions of the chromosome.
Deep sequencing methods allow the population of siRNAs in a cell to be mapped
onto their regions of homology. The abundance of small RNAs and
transposons/retrotransposons along each of the five Arabidopsis chromosomes is
shown relative to the centromere (circle on each line representing the
chromosomes). In Arabidopsis these repetitive elements occur primarily in
pericentromeric regions.
Transposons and repetitive elements are more dispersed in organisms with larger
genomes.
45
46
Pro35S: KAN Pro35S: HYG
x
Expected Results
Selection on kanamycin only: 50% KanR
Selection on hygromycin only: 50% HygR
Selection on Kan + Hyg: 25% KanR and HygR
Transcriptional gene silencing
Transcriptional silencing on the example of crossing of two lines of transgenic
plants, each of them being heterozygous (hemizygous) for a single copy transgene
with different resistance marker.
The green ovals represent cotyledons of healthy, antibiotic resistant seedlings. The
parental lines carry one copy of the transgene which acts as a dominant trait.
Therefore, 50% of the progeny from the cross should carry any one dominant trait,
and 25% should carry both. Note that these transgenes insert at different loci – they
are independent. They are not two alleles of a single locus.
46
47
Pro35S : HYG
Observed Results
Selection on kanamycin only: 50% KanR
Selection on hygromycin only: 0% HygR
Selection on Kan + Hyg: 0% KanR and HygR
Pro35S : KAN
Transcriptional gene silencing
x
Sometimes one of the transgenes was silenced in the progeny carrying both genes.
That is reflected in the observed ratios of observed frequency of plant resistant to
both on of the used antibiotics. In this case, the gene for resistance to hygromycin
was silenced.
47
48
CaMV 35S pro :
HYG
CaMV 35S pro : HYG
The promoter of the silenced gene
become methylated, interfering thus
with transcription.
Pro35S : HYG
DNA methylation
Pro35S : KAN
Transcriptional gene silencing
The mechanism of silencing in this case is promoter methylation.
48
49
 The siRNA pathway silences foreign DNA, transposons and repetitive
elements.
 In plants, siRNAs are produced by the action of Dicer-like proteins
dicing dsRNA into 24 nt siRNAs
 The siRNAs associate with AGO proteins and form silencing
complexes
 The silencing complexes can act post-transcriptionally on RNA
targets, cleaving them or interfering with translation
 The silencing complexes can also act on chromatin, silencing their
targets by DNA methylation or histone modification
siRNAs - summary
50
Outline of Lesson 10
Regulation of Gene Expression during Development
 Overview of levels of gene expression regulation
 Transcriptional gene regulation
 Modification of the chromatin structure and DNA methylation
 Transcriptional activation
 Post-transcriptional gene regulation
 Splicing of hnRNA
 Translation initiation
 Localization of mRNA
 Protein localization
 RNA interference
 Identification and mechanism of gene expression regulation
via RNA interference
 siRNA-mediated silencing
 miRNA-mediated silencing
50
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AAAn
RNA Pol II
MIR gene
RNA Pol II
mRNA
AGO
AGO
AAAn
AAAn
AGO
Translational
interference
mRNA
slicing
Mechanisms of miRNAs action
miRNAs in plants
• small # of highly conserved miRNAs
• hing # of non-conserved miRNAs
• binding to 5’UTR and require almost complete complementarity
• most of the plant miRNA induce slicing of target mRNAs
microRNAs slice mRNAs or interfere with their translation. Thus, in contrast to
siRNAs, miRNAs do not induce transcriptional silencing.
miRNAs are thought to have evolved from siRNAs, and are produced and
processed somewhat similarly. Plants have a small number of highly conserved
miRNAs, and a large number of non-conserved miRNAs.
miRNAs are encoded by specific MIR genes but act on other genes – they are
trans-acting regulatory factors. miRNAs in plants regulate developmental and
physiological events.
Plant miRNA bind preferentially to the 5’UTR or coding sequence and require almost
complete complementarity with their targets.
In contrast to that, in animals there is much higher proportion of genes being
regulated by miRNAs. The most of animal miRNA bind to the 3’UTR and extensive
sequence complementarity is not required.
In animals, dominant regulatory mechanism mediated by miRNAs is translational
repression, while in plants, most of the miRNAs initiate slicing of target mRNAs.
51
52
Reprinted from Margis, R., Fusaro, A.F., Smith, N.A., Curtin, S.J., Watson, J.M., Finnegan, E.J., and Waterhouse, P.M.
(2006) The evolution and diversification of Dicers in plants FEBS Lett. 580: 2442-2450 with permission from Elsevier.
AtDCL1 produces miRNA
AtDCL2 - 4 produce siRNA
miRNAs and siRNAs are processed by related but different
DCL proteins
Plants have 4 or more DCL proteins, more than found in other organisms. The
amplification of DCL proteins is thought to allow plants great flexibility in pathogen
defence responses.
Note that mammals make do with one dicer, and insects and fungi with two. Like
most components of the siRNA pathway, dicer-like genes are amplified in plants.
52
53
AGO1
AGO4
AGO1 preferentially slices
its targets and associates
with miRNAs but also
some siRNAs
AGO4 preferentially
associates with siRNA
and mediates methylation
of source DNA.
Reprinted from Vaucheret, H. (2008) Plant ARGONAUTES. Trends Plant Sci. 13: 350-358 with permission from Elsevier.
miRNAs and siRNAs associate with several AGO proteins
miRNAs in plants
• small # of highly conserved miRNAs
• hing # of non-conserved miRNAs
Arabidopsis has 10 AGO proteins. They are not all well characterized and there is
some functional overlap.
53
54
3'
5' miRNA
miRNA*
3'
5' pri-miRNA
miRNA
MIR gene
mRNA target
MIR genes are transcribed into long RNAs that are processed
to miRNAs
Primary miRNA
DCL1 processing and
miRNA-miRNA* duplex
formation
Transport irom nucleus
into the cytoplasm and
miRNA* degrdation
miRNAs are encoded by MIR genes. MIR genes are transcribed into long RNAs
called primary miRNA (pri-miRNA) with partial complementarity.
The pri-miRNA transcript folds back into a partially double-stranded stem-loop or
hairpin structure, which is processed by DCL1. The product of DCL cleavage dsRNA
with a 2-nucleotide 3´ overhang is called a miRNA-miRNA* duplex.
The miRNA-miRNA* duplex is transported from the nucleus into the cytoplasm by
the homologue of exportin 5 HASTY (will be discussed later).
Subsequently, the miRNA* strand is degraded.
54
55
Fahlgren, et al., PLoS ONE, 2007
•Non-conserved MIRNA families
usually occur as single genes
•Conserved ones have often
duplicated to larger gene families
Factors
Some miRNAs are highly conserved and important gene
regulators
In spite of there are quite few genes regulated by miRNAs in plants, there is high
number of MIR genes.
However, most of them are evolutionary non-conserved and probably provide no
competitive advantage. It has been suggested that there is high rate of birth and
death and only few of them became stabilized in the genome.
Nearly half of the targets of conserved miRNAs are transcription factors.
55
56
Reprinted from Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and Bartel, D.P. (2002) MicroRNAs in plants. Genes Dev. 16: 1616–1626.
The MIR156 gene family is highly conserved
Arabidopsis miR156 gene family
miRNA*
Among the evolutionary conserved, i.e. important regulators belong miRNAs that
target TF. E.g. miR156 family is highly conserved within the plant kingdom (it is
found in angiosperms as well as mosses)
miR156 is encoded by six or more genes in Arabidopsis and targets transcription
factors that control developmental phase changes (see further slides).
The red sequence indicates the miRNA produced from each of the six Arabidopsis
MIR156 genes (MIR156A – MIR156F). The boxed sequence shows the miRNA*.
56
57
miRNA gene family Target gene family Function
156 SPL transcription factors Developmental timing
160 ARF transcription factors Auxin response,
development
165 HD-ZIPIII transcription
factors
Development, polarity
172 AP2 transcription factors Developmental timing, floral
organ identity
390 TAS3 (tasiRNA) which acts
on ARF transcription factors
Auxin response,
development
395 Sulfate transporter Sulfate uptake
399 Protein ubiquitination Phosphate uptake
Adapted from Willmann, M.R., and Poethig, R.S. (2007) Conservation and evolution of miRNA regulatory programs
in plant development. Curr. Opin. Plant Biol. 10: 503–511..
Targets of some conserved miRNAs
The table shows targets of some of the evolutionary conserved MIR genes (or
rather respective miRNAs). As discussed previously, these are mostly MIR genes
encoding miRNAs recognizing TFs.
57
58
Reprinted from Willmann, M.R., and Poethig, R.S. (2007) Conservation and evolution of miRNA regulatory
programs in plant development. Curr. Opin. Plant Biol. 10: 503–511 with permission from Elsevier.
Gene duplication
Plant miRNAs are thought to be distantly related to their
targets
Plant miRNAs are thought to be derived from their target sequences following gene
duplication, inverted duplication and divergence.
Only some miRNAs confer selective advantage and are retained and further duplicated.
58
59
Germination
zygote
JUVENILE
PHASE
Vegetative phase
change
ADULT
PHASE
REPRODUCTIVE
PHASE
EMBRYONIC
PHASE
miRNAs and vegetative phase change
Vegetative phase change is the transition from juvenile to adult growth in plants.
The transition includes changes in the leaf shape and arrangenment, internode
length, and the accumulation of epidermal hairs (trichomes) and waxes.
59
60
Photos courtesy of James Mauseth
Adult
Juvenile
A
J
Vegetative phase change affects morphology and
reproductive competence
Some cacti have very different juvenile and adult growth patterns.
60
61
Eucalyptus globulus
Juvenile leaves:
rounded,
symmetrical,
opposite
phyllotaxy
Adult leaves:
elongated,
asymmetrical,
alternating
phyllotaxy
Phase change can affect leaf shape, phyllotaxy, and trichome
patterns
Eucalyptus leaves are strongly dimorphic, as are leaves of holly and ivy. In other
plants including Arabidopsis and maize the change is more subtle.
61
62
Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier.
In Arabidopsis, phase change affects leaf shape and trichome
patterning
In Arabidopsis, juvenile leaves are rounder, less serrated, and have trichomes only
on the upper (adaxial) surface; adult leaves also have trichomes on the lower
(abaxial) surface.
In the next few slides the leaves are colour coded as juvenile (olive), adult (green)
or reproductive, also sometimes called cauline leaves (red) based on their
morphology and anatomy. The boxes above the leaves schematically indicate the
relative duration of each phase (for comparing wild-type and mutant plants).
62
63
Reprinted with permission from Bollman, K.M. Aukerman, M.J., Park, M.-Y., Hunter, C., Berardini, T.Z., and Poethig, R.S. (2003) HASTY, the
Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development 130: 1493-1504.
hasty
Wild-type
Phase change is specified by miRNAs
miRNA export from
nucleus
HASTY, with a shortened juvenile phase, encodes a protein needed for miRNA
export from nucleus to cytoplasm.
It’s easy to see from these figures that hasty has a shortened juvenile phase relative
to wild-type.
63
64
Reprinted from Hunter, C., Sun, H., and Poethig, R.S. (2003) The Arabidopsis heterochronic gene ZIPPY is
an ARGONAUTE family member. Curr. Biol. 13: 1734–1739, with permission from Elsevier.
Wild-type
zippy
Phase change is specified by miRNAs
AGO7
Loss-of-function zippy mutants prematurely express adult vegetative traits. ZIPPY
encodes an ARGONAUTE protein, AGO7.
Zippy prematurely expresses adult traits; this is a different effect that hasty, in which
the total non-reproductive phase was shortened as well.
64
65
Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier.
miR156 overexpression prolongs juvenile phase in
Arabidopsis
As could be clearly seen from the phenotyope of transgenic line with ectopic
overexpression of miR156 gene, there is dramatic elongation of the juvenile phase.
Highly conserved miR156 targets SQUAMOSA PROMOTER BINDING PROTEINbox
(SBP-box) genes in maize, called SPL genes in Arabidopsis.
SBP-box genes are TFs that regulate expression of key regulators of flowering TF,
SOC1, LFY, and AP1 and thus regulate the transition from juvenile to
adult/reproductive growth phase.
65
66
miR156
binding
site
miR156 SPL
ORF 3’ UTRPromoterSPL3
miR156 targets SQUAMOSA PROMOTER BINDING PROTEINLIKE
(SPL) genes, promoters of phase change
SBP-box TF
The SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes are a family
of SBP-box transcription factors that are miR156 targets.
In wild-type plants, miR156 expression decreases with plant age, allowing SPL to
accumulate and promote phase change.
The graph indicates the relative activity of miR156 and SPL, showing that as
miR156 levels decrease SPL levels increase. When miR156 levels are high, SPL
mRNA is cleaved and the protein can’t accumulate.
66
67
Reproduced with permission from Wu, G., and Poethig, R.S. (2006) Temporal regulation of shoot development in Arabidopsis
thaliana by miR156 and its target SPL3. Development 133: 3539–3547.
ORF 3’ UTRPromoter
ORFPromoter
ORF 3’ UTRPromoter
SPL3
SPL3Δ
SPL3m
miR156
binding
site
Wild-
type
miR156-
resistant
miR156 SPL
miR156 SPL
miR156-resistant SPL promotes precocious phase change
The miR156 effect can be eliminated by deletion of the 3’ UTR or mutations that
interfere with miR156 binding, as shown schematically.
In either case, SPL protein accumulates early (it is indifferent to the presence of
mir156), promoting phase change. Note that the miR156 resistant plants are
flowering when the control plants are still vegetative.
67
68
Reprinted from Poethig, R.S. (2009) Small RNAs and developmental timing in plants. Curr. Opin. Genet. Devel. 19: 374-378, with permission from Elsevier.
Wild-
type
miR156-
loss-of-
function
miR156
OE
miR156 SPL
SPL
miR156 loss-of-function promotes precocious phase change
Similarly, interfering with miR156 production allows SPL to accumulate early and
promotes precocious phase change.
68
69
Aukerman, M.J., and Sakai, H. (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-Like target genes Plant Cell
15: 2730-2741.
miR156 SPL
miR172 glossy15
In Arabidopsis, SPL9
directly activates
transcription of
MIR172c
Arabidopsis plants
overexpressing miR172 flower
early.
Wild-type miR172-OX
Phase change involves a temporal cascade of miRNAs and
transcription factors
Interestingly, in Arabidopsis, MIR172 gene is induced by one of the SPL
transcription factors, suggesting that a cascade of miRNA-regulated genes controls
phase transition.
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lin-14 mRNA
lin-4 miRNA
lin-14 gene
3’ untranslated region
lin-4 binding sites
Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:
843–845. Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C.
elegans. Cell 75: 855–862.
miRNAs regulate developmental timing in other organisms
Proper larval
development
miRNAs were discovered in studies of developmental progressions in the nematode
C. elegans. miRNA encoded by lin-4 is required for proper larval development.
Genes lin-14 and lin-4 promote or repress developmental progression in the
nematode C. elegans. Genetic studies showed that lin-4 acts antagonistically to lin-
14 but that lin-4 does not encode any protein.
The functional domain of lin-4 was identified and found to be complementary to
several regions in the 3´ UTR of lin-14 (see the figure, red).
Subsequently, the interaction between these complementary sequences was shown
to be necessary for lin-4’s repression of lin-14, revealing the basis for gene
regulation by miRNAs.
70
71
Wild-type C. elegans
lin-4 Loss-of-function lin-4 is a negative
regulator of lin-14.
In wild-type worms,
lin-14 is expressed
early and then shut
off.
lin-14
expressio
n
lin-4 loss-of-
function
causes lin-14
expression to
remain high.
Lee, R.C., Feinbaum, R.L., and Ambrose, V. (1993). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:
843–845. Wightman, B., Ha, I., and Ruvkun, G. (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C.
elegans. Cell 75: 855–862.
Downregulation of lin-14 by lin-4 is necessary for normal
development
For proper developmental progression in C. elegans, the juvenile-promoting activity
of the lin-14 gene has to be repressed by the action of the miRNA encoded by lin-4.
71
72
Vegetative phase change affects morphology and reproductive
competence
miRNAs contribute to the temporal control of gene expression and
phase change
miR156 promotes juvenile phase by preventing SPL gene
accumulation
SPL genes promote phase change and flowering
In Arabidopsis, a SPL protein promotes transcription of miR172
mir172 triggers phase change by interfering with GLOSSY15
expression
In the nematode C. elegans, lin-4 silencing of lin-14 is required for
developmental progression
miRNAs and phase change - summary
73
Key Concepts
Regulation of Gene Expression during Development
□ Regulation of gene expression occurs at different levels,
from transcriptional till the posttranscriptional and
posttranslational
□ Basal promoters are co-regulated in a combinatorial way via
spectrum of positive and negative factors
□ mRNA and protein localizations belong to the most important
posttranscriptional regulations of gene expression
□ RNA interference is natural and powerful mechanism allowing
regulation of gene expression at both transcriptional and
posttranscriptional levels
□ dsRNA is either trigger or intermediate in the RNAi-mediated
regulation
□ siRNA and miRNA are two major effector molecules
regulating different and complementary spectrum of target
genes
73
74
74
Discussion