This document summarizes transcriptional gene regulation in eukaryotes. It discusses the basic mechanisms of transcription including initiation, elongation, and termination. It describes the role of general transcription factors, promoter elements like TATA boxes, and RNA polymerase in basal transcription. It also explains how distal enhancer elements and transcription factors regulate gene expression by interacting with the promoter and basal transcription machinery. Chromatin structure is also noted to influence transcription. The modular and combinatorial nature of transcriptional control is emphasized.
Regulation of gene expression in eukaryotesAnna Purna
Presence of nucleus and complexity of eukaryotic organism demands a well controlled gene regulation in eukaryotic cell. Tissue specific gene expression is essential as they are multicellular organisms in which different cells perform different functions. This PPT deals with various control points for the gene regulation and expression within a cell.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
Replication,transcription,translation complete the central dogma of life.How mRNA,tRNA,rRNA act on ribosomes for protein synthesis.Difference between eukaryotes and prokaryotes
Regulation of gene expression in eukaryotesAnna Purna
Presence of nucleus and complexity of eukaryotic organism demands a well controlled gene regulation in eukaryotic cell. Tissue specific gene expression is essential as they are multicellular organisms in which different cells perform different functions. This PPT deals with various control points for the gene regulation and expression within a cell.
REGULATION OF
GENE EXPRESSION
IN PROKARYOTES & EUKARYOTES .
This presentation is enriched with lots of information of gene expression with many pictures so that anyone can understand gene expression easily.
Gene expression is the process by which the information encoded in a gene is used to direct the assembly of a protein molecule.
Gene expression is explored through a study of protein structure and function, transcription and translation, differentiation and stem cells.
It is the process by which information from a gene is used in the synthesis of a functional gene product.
These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea)
Regulation of gene expression:
Regulation of gene expression includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
Gene regulation is essential for viruses, prokaryotes and eukaryotes as it increases the versatility and adaptability of an organism by allowing the cell to express protein when needed.
CLASSIFICATION OF GENE WITH RESPECT TO THEIR EXPRESSION:
Constitutive ( house keeping) genes:
Are expressed at a fixed rate, irrespective to the cell condition.
Their structure is simpler.
Controllable genes:
Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
Their structure is relatively complicated with some response elements.
TYPES OF REGULATION OF GENE:
positive & negative regulation.
Steps involving gene regulation of prokaryotes & eukaryotes.
Operon-structure,classification of mechanisms- lac operon,tryptophan operon ,
and many things related to gene expression.
This is a video slide so anyone can understand this topic easily by seeing pictures included in this slide.
Replication,transcription,translation complete the central dogma of life.How mRNA,tRNA,rRNA act on ribosomes for protein synthesis.Difference between eukaryotes and prokaryotes
Control of gene expression ppt
definition of gene expression
inducible gene expression
repressible gene expression
control of gene expression in eukaryotics .all the in information about this topic is include .
1. Levels of gene regulation
The observation that differences in the RNA and protein content of different tissues are not paralleled by significant differences in their DNA content indicates that the process whereby DNA produces mRNA must be the level at which gene expression is regulated in eukaryotes. In bacteria this process involves only a single stage, that of transcription, in which RNA copy of the DNA is produced by the enzyme RNA polymerase. Even while this process is still occurring, ribosomes attach to the nascent RNA chain and begin to translate it into protein. Hence cases
of gene regulation in bacteria, such as the switching on of the synthesis of the enzyme β-galactosidase in response to the presence of lactose (its substrate), are mediated by increased transcription of the appropriate gene. Clearly, a similar regulation of gene transcription in different tissues, or in response to substances such as steroid hormones which induce the synthesis of new proteins, represents an attractive method of gene regulation in eukaryotes.
In contrast to the situation in bacteria, however, a number of stages intervene between the initial synthesis of the primary RNA transcript and the eventual production of mRNA (Fig. 1).
The initial transcript is modified at its 5′ end by the addition of a cap structure containing a modified guanosine residue and is subsequently cleaved near its 3′ end, followed by the addition of up to 200 adenosine residues in a process known as polyadenylation. Subsequently, intervening sequences or introns, which interrupt the protein-coding sequence in both the DNA and the primary transcript of many genes. Although this produces a functional mRNA, the spliced molecule must then be transported from the nucleus, where these processes occur, to the cytoplasm where it can be translated into protein.
Basics of Undergraduate/university fellows
Transcription is more complicated in eukaryotes than in prokaryotes because
eukaryotes possess three different classes of RNA polymerases and because of the
way in which transcripts are processed to their functional forms.
More proteins and transcription factors are involved in eukaryotic transcription.
Control of gene expression ppt
definition of gene expression
inducible gene expression
repressible gene expression
control of gene expression in eukaryotics .all the in information about this topic is include .
1. Levels of gene regulation
The observation that differences in the RNA and protein content of different tissues are not paralleled by significant differences in their DNA content indicates that the process whereby DNA produces mRNA must be the level at which gene expression is regulated in eukaryotes. In bacteria this process involves only a single stage, that of transcription, in which RNA copy of the DNA is produced by the enzyme RNA polymerase. Even while this process is still occurring, ribosomes attach to the nascent RNA chain and begin to translate it into protein. Hence cases
of gene regulation in bacteria, such as the switching on of the synthesis of the enzyme β-galactosidase in response to the presence of lactose (its substrate), are mediated by increased transcription of the appropriate gene. Clearly, a similar regulation of gene transcription in different tissues, or in response to substances such as steroid hormones which induce the synthesis of new proteins, represents an attractive method of gene regulation in eukaryotes.
In contrast to the situation in bacteria, however, a number of stages intervene between the initial synthesis of the primary RNA transcript and the eventual production of mRNA (Fig. 1).
The initial transcript is modified at its 5′ end by the addition of a cap structure containing a modified guanosine residue and is subsequently cleaved near its 3′ end, followed by the addition of up to 200 adenosine residues in a process known as polyadenylation. Subsequently, intervening sequences or introns, which interrupt the protein-coding sequence in both the DNA and the primary transcript of many genes. Although this produces a functional mRNA, the spliced molecule must then be transported from the nucleus, where these processes occur, to the cytoplasm where it can be translated into protein.
Basics of Undergraduate/university fellows
Transcription is more complicated in eukaryotes than in prokaryotes because
eukaryotes possess three different classes of RNA polymerases and because of the
way in which transcripts are processed to their functional forms.
More proteins and transcription factors are involved in eukaryotic transcription.
Eukaryotic transcription is the elaborate process that eukaryotic cells use to copy genetic information stored in DNA into units of RNA replica.- Source: Wikipedia
Transcription in eukaryotes: A brief view
Transcription is the process by which single stranded RNA is synthesized by double stranded DNA. Transcription in eukaryotes and prokaryotes has many similarities while at the same time both showing their individual characteristics due to the differences in organization. RNA Polymerase (RNAP or RNA Pol) is different in prokaryotes and eukaryotes. Coupled transcription is seen in prokaryotes but not in Eukaryotes. In eukaryotes the pre-RNA should be spliced first to be translated.
In Eukaryotic transcription, synthesis of RNA occurs in the 3’→5’ direction. The 3’ end is more reactive due to the hydroxide group. 5’ end containing phosphate groups meanwhile, is not very reactive when it comes to adding new nucleotides. In Eukaryotes, the whole genome is not transcribed at once. Only a part of the genome is transcribed which also acts as the first, principle stage of genetic regulation.
Eukaryotes have five nuclear polymerases:
• RNA Polymerase I: This produces rRNA (23S, 5.8S, and 18S) which are the major components in a ribosome. This also produces pre-rRNA in yeasts.
• RNA Polymerase II: Helps in the production of mRNA (messenger RNA), snRNA (small, nuclear RNA), miRNA. This is the most studied type and requires several transcription factors for its binding
• RNA Polymerase III: This synthesizes tRNA (transfer RNA), 5S rRNA and other small RNAs required in the cytosol and nucleus.
• RNA Polymerase IV: Synthesizes siRNA (small interfering RNA) in plants.
• RNA Polymerase V: This is the least studied polymerase and synthesizes siRNA-directed heterochromatin in plants.
Eukaryotic transcription can be broadly divided into 4 stages:
• Pre-Initiation
• Initiation
• Elongation
• Termination
Transcription is an elaborate process which cells use to copy the genetic information stored in DNA into RNA. This pre-RNA is modified into mRNA before being transcribed to proteins. Transcription is the first step to utilizing the genetic information in a cell. Both Eukaryotes and Prokaryotes employ this process with the basic phases remaining the same. However eukaryotic transcription is more complex indicating the changes transcription has undergone towards perfection during evolution.
As a periodontist, it is of utmost importance to understand the genetic basis of inheritance in periodontal diseases be able to relate to the various polymorphisms associated with periodontal diseases. This ppt presents the basics of genetics from the point of view of future understanding of polymorphisms related to periodontal diseases.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Let's dive deeper into the world of ODC! Ricardo Alves (OutSystems) will join us to tell all about the new Data Fabric. After that, Sezen de Bruijn (OutSystems) will get into the details on how to best design a sturdy architecture within ODC.
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Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
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Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
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https://arxiv.org/abs/2306.08302
2. Microsoft Research's GraphRAG paper and a review paper on various uses of knowledge graphs:
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Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
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Length: 30 minutes
Session Overview
-------------------------------------------
During this webinar, we will cover the following topics while demonstrating the integrations of JMeter, InfluxDB and Grafana:
- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
- What are the benefits of integrating InfluxDB and Grafana into the load testing stack?
- Which features are provided by Grafana?
- Demonstration of InfluxDB and Grafana using a practice web application
To view the webinar recording, go to:
https://www.rttsweb.com/jmeter-integration-webinar
7. Transcription
Initiation, elongation, termination
Catalyzed by RNA polymerase
– “Transcription bubble”: DNA transiently separated into
single strands
– One strand is used as a template
– Unwinding point & rewinding point
– Rate ≈ 40 nucleotides/second at 37° for bacteria
RNA polymerase
– Many subunits: catalytic site, CTD with (YSPTSPS)n
– pol I, pol II, pol III
14. Basal transcription apparatus (general
factors & RNA polymerase)
Proximal cis-regulatory module
Distal cis-regulatory modules
Modules = discrete DNA elements that contain specific
sequence motifs with which DNA binding proteins interact
and transmit molecular signals to genes
Promoter
Enhancer
15. BTA
– General factors: TFIIx
– Mechanics of initiating RNA
synthesis at all promoters
– Determines location of
transcription startpoint
– Complex with RNA polymerase
– TATA
• ~ 25bp upstream
• 8bp consensus of A•T pairs
• Tends to be surrounded by
G•C rich regions
• TBP, 11 TAFs : TFIID (~800kD)
18. Promoter
recognition Function = to be recognized by proteins; so differs
from exon, …
Any essential nucleotide sequence should be
conserved
– Some variation is permitted
– When is it sufficiently conserved?
– Idealized sequence with base most often present:
consensus sequence by aligning all known examples
– Only conservation of very short sequences; 60 bp
associated with RNA pol lack conservation
19. Variety of elements can contribute, none is essential
for all promoters (mix & match principle)
CAAT box ~ -80bp GGCCAATCT
– increases promoter strength
– Bound by CTF/NF1 family, CP1 & CP2, C/EBP, ACF
GC box GGGCGG
– SP1
Octamer (8bp) ATTTGCAT
– Bound by Oct1 (ubiquitous): activates histon H2B
– Bound by Oct2 (lymphoid cells): Ig kappa light chain
– context is important
20. Modular nature of the promoter:
– Equivalent regions can be exchanged
– Main purpose = to bring the factors they bind into the
vicinity of the initiation complex
– Protein-protein interactions determine the efficiency of
the initiation reaction
Sequence elements influence the frequency of
initiation
Repression of transcription:
– Generally by influencing chromatin structure
– By repressors, e.g. Dr1/DRAP1 binds to TBP and
CAAT displacement protein (CDP)
23. Modules
50 bp to 1.5 kbp in size
4-8 TFs (often multiple sites); higher density of regulatory
elements than in the promoter
Many elements are common elements in promoters, e.g.
AP1 and the octamer
Can stimulate any promoter placed in its vicinity
Can function anywhere (cfr β-globin: 200 fold in vivo) ;
Position relative to promoter can vary substantially; can
function in either orientation
24.
25. Binding sites for activators that control transcription of the mouse transthyretin (TTR)
promoter in hepatocytes. HNF = hepatocyte nuclear factor. [See R. Costa et al., 1989, Mol. Cell
Biol. 9:1415; K. Xanthopoulus et al., 1989,Proc. Nat’l. Acad. Sci. USA 86:4117.]
Example: TTR
26. Example: muscle specific modules
Creatine kinase, myosin light chain, skeletal actin, myosin heavy chain
27. Example: β-globin
Model for the control of the human β-globin gene. Some of the gene regulatory proteins shown,
such as CP1, are found in many types of cells, while others, such as GATA-1, are present in only
a few types of cells including red blood cells and therefore are thought to contribute to the cell-
type specificity of β -globin gene expression. (Adapted from B. Emerson, In Gene Expression:
General and Cell-Type-Specific [M. Karin, ed.], pp. 116-161. Boston: Birkhauser, 1993.)
30. Current view:
– same sort of interaction with basal apparatus as the
proximal promoter module
– Increase the concentration of transcription factors in the
vicinity of the promoter
Intervening DNA: extruded as a large “loop”
Generality: not yet clear (what proportion of
promoters require an enhancer?)
31. Four activators enriched in
hepatocytes plus the ubiquitous
AP1 factor bind to sites in the
hepatocytespecific enhancer and
promoter-proximal region of the
TTR gene.
The activation domains of the
bound activators interact
extensively with co-activators,
TAF subunits of TFIID,
Srb/Mediator proteins, and
general transcription factors,
resulting in looping of the DNA
and formation of a stable
activated initiation complex.
Cooperative assembly
32. Limited knowledge
Experimentally verified binding sites
Experimentally verified “composite elements” or
CE’s
– GR site + AP-1 in proliferin promoter
– Synergistic: result in non-additively high level
– Antagonistic: overlapping sites, masking an activation
domain,…
– Direct or through coactivator
Few modules characterized that have multiple
elements, some in developmental biology
33. Side-track: Transcription factors
5% of our proteins
Activities controlled in regulatory pathways
Independent domains responsible for activities:
– Recognition of specific target sequences
– Binding to other components
of the transcription apparatus
– E.g. yeast GAL4
34. Protein-DNA interactions
– Proteins with high affinity for a specific sequence also
possess a low affinity for any (random) DNA sequence
– E.g. Lac repressor E. coli: Free:bound = 10-4
– High-affinity site competes with the large number of
low-affinity sites; repressor binds ≈107
times better to
operator DNA (bound 96% of time for 10
molecules/cell)
35. How the different base
pairs in DNA can be
recognized from their
edges without the need to
open the double helix.
36. The binding of a gene regulatory protein to the major
groove of DNA.
Typically, a protein-DNA interface
consists of 10 to 20 such contacts,
involving different amino acids, each
contributing to the binding energy of
the protein-DNA interaction.
38. All of the proteins bind DNA as dimers in which the two copies of the recognition helix (red
cylinder) are separated by exactly one turn of the DNA helix (3.4 nm). The second helix of the
helix-turn-helix motif is colored blue. The lambda repressor and cro proteins control
bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite
activator protein (CAP) control the expression of sets of E. coli genes.
Helix-Turn-Helix
39. Homeodomains
– Related to helix-turn-helix bacterial repressors
– Homeobox = 60 AA residues
– E.g. en, eve, Hox, Oct-1, Oct-2 (Oct also have Pou domain
next to homeodomain)
The homeodomain is folded into three alfa helices, which are packed tightly together by hydrophobic interactions (A). The part
containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts
with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor
groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein, but it is
nearly identical to two homeodomains from Drosophila, which interact with DNA in a similar fashion. (Adapted from C.
40. Helix-loop-helix (HLH)
– DNA binding (helix) & dimerization
– Class A: ubiquitouslyh expressed proteins, e.g.
E12/E47
– Class B: tissue-specific expression, e.g. MyoD,
myogenin, Myf-5
– Myc proteins (separate class)
Leucine zippers fig 21.15
– Dimerization motif
– E.g. Jun+Fos = AP1
– Gcn4 ->
42. Figure 1 Genome-wide comparison of transcriptional activator families in eukaryotes.
The relative sizes of transcriptional activator families among Homo sapiens,
D. melanogaster, C. elegans and S. cerevisiae are indicated, derived from an analysis of
eukaryotic proteomes using the INTERPRO database, which incorporates Pfam, PRINTS
and Prosite. The transcription factors families shown are the largest of their category out
of the 1,502 human protein families listed by the IPI.
44. Transcription factories
cfr. replication factories
Active RNA polymerases are concentrated in discrete
'factories' where they work together on many different
templates
Complexes for transcription and RNA processing are
likely to be immobile structures within the gel-like
nucleoplasm (Burns et al, 2001; Kimura et al, 1999)
Transcriptional interference: phenomenon where
transcription of one gene prevents transcription of an
adjacent gene. Discovery: Cells were transfected with a
retroviral vector encoding resistance to neomycin and
azaguanine, and clones harboring a single copy of the
vector selected. Expression of the 3' gene was
suppressed when selection required expression of the 5'
gene, and vice versa. In addition, hardly any cells grew
in both neomycin and azaguanine
45. Cook, 1999 (Science)• Enhancers
•dynamic equilibrium
•enhancing the probability of the key transcription cycle interactions
•Element 5’ or 3’ doesn’t matter!
46. Recap: evolution of understanding of
eukaryotic transcription
Lemon and Tjian, Genes Dev. 14: 2551-2569 (2000)
49. Activate/inactivate a TF
Transport through nuclear pores from cytoplasm
to nucleus (e.g. masking NLS, nuclear localization
signal, can regulate this transport)
Link to Ubiquitin protease system
– Rapid turnover of promoter bound TF: resets signaling
pathway: cell can continuously monitor its environment
Tissue-specific synthesis
– Development, e.g. homeodomain proteins
Modification
– Phosphorylation, acetylation, methylation
– E.g., AP1 (= Jun+Fos) → active form by
phosphorylation
– E.g., p53 acetylated (modulates interactions with
coactivator and repressor proteins
50. Ligand binding
– E.g. Steroid receptors
– Influence: localization or DNA-binding ability
Cleavage
Inhibitor release
– E.g. NF-κB + I- κB (release in B lymphocytes)
Change of partner (active partner displaces
inactive partner)
53. Level 1 = active/inactive factor
Level 2 = cooperation of multiple factors within a
module (all present and active, and all repressors
inactive or absent)
Level 3 = multiple autonomous modules per gene
– Each module can independently activate the gene
– Each has a specific function (e.g. activation in certain
cell type or at particular stage in dvl)
– different circuits of regulation, e.g. metallothionein
gene (MT): heavy metals and steroids, fig 21.1
– Gene can respond to multiple signaling pathways
– Facilitates fine-tuning of transcript levels
54. Combinatorial and context dependent
regulation of transcription
– one factor can induce transcription of one gene
while repressing that of another
55. Example: eve
Experiment demonstrating the modular
construction of the eve gene regulatory region.
(A) A 480-nucleotide-pair piece of the eve
regulatory region was removed and inserted
upstream of a test promoter that directs the
synthesis of the enzyme β-galactosidase (the
product of the E. coli lacZ gene). (B) When this
artificial construct was reintroduced into the
genome of Drosophila embryos, the embryos
expressed β-galactosidase (detectable by histo-
chemical staining) precisely in the position of the
second of the seven eve stripes (C).
(Metamerization)
-
+
57. Principles for specification
1. cis-regulatory transformation of input patterns into
spatial domains of differential gene expression
2. Always assemblages of diverse target sites because
multiple inputs are required
3. Output=novel with respect to any one of the incident
inputs + more precise in space and time => “information
processing”
4. Every specific type of interaction that can be detected in
vitro is fundamentally significant (it is unlikely that highly
specific site clusters, which are of improbable random
occurrence would have no function)
5. Negative & positive inputs
(Davidson, 2001)
58. Cis-regulatory logic device
endo16 of Strongylocentrotus (zee-egel)
Secreted embryonic gut protein
“hardwired biological computational
device”
59.
60.
61.
62. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Influence of chromatin structure
Oncogenes
Techniques
63. Chromatin
Eukaryotic genomes are
packaged with chromatin
proteins
Heterochromatin (highly
condensed, untranscribed)
Euchromatin (more
accessible, transcribed)
Each cell: unique pattern of
heterochromatin and
euchromatin
65. Chicken and egg scenario
TF binding requires chromatin decompaction by
certain factors but the latter also need to interact
with DNA
Solution: probably some TFs can bind to their
recognition sequences even when they are
packaged (e.g. glucocorticoid receptor: only
contacts DNA on one side ⇔ NF1 surrounds
double helix)
67. 2. Histone-modifying complexes
• Phosphorylation, methylation, acetylation
• Histone acetyltransferase (HAT), histone
deacetylase (HDAC)
• How do they impact the structure of the template
and the ability of the transcription machinery to
function?
• lowered positive charge on acetylated N termini,
lowered stability of interaction with DNA
• Disrupting internucleosomal interactions
• Recruiting additional TFs
• A lot of combinatorial possibilities: histon code?
69. Model of the protein
interactions and functions
of the Myc/Max/Mad
transcription network.
Myc-Max and Mad-Max (along with Mnt-Max and Mga-Max) complexes bind to DNA to E-boxes. Binding can be
affected by the context, sequence, cooperativity, and location of the E-boxes. Myc-Max heterodimers activate
transcription by recruiting HAT's via TRRAP. This leads to the acetylation of histone tails and the opening of local
chromatin structure. Additionally, Myc-Max appears to repress transcription through Inr elements via an undefined
mechanism. As a result of these activities at target genes, Myc affects proliferation, cell cycle, growth, immortalization,
and apoptosis. When deregulated, Myc cooperates with other oncogenes to cause a variety of cancers.
Mad-Max and Mnt-Max heterodimers repress transcription by recruiting HDAC's via mSin3A. This leads to the
deacetylation of histone tails and the closing of local chromatin structure. As a result of target gene repression, Mad
causes an increased cell doubling time, growth arrest, and the maintenance of differentiation.
Grandori C, Cowley SM, James LP,
Eisenman RN.
Annu Rev Cell Dev Biol. 2000;16:653-
99
70. Cytosine methylation
mCG often in inactive vertebrate genes
After replication of methylated DNA,
methyl groups are added to daughter strands
CpG islands
imprinting
71. Imprinting
Imprinted genes are genes whose expression is determined by the
parent that contributed them.
Imprinted genes violate the usual rule of inheritance that both alleles in
a heterozygote are equally expressed.
Examples of the usual rule:
– If a child inherits the gene for blood group A from either parent and the
gene for group B from the other parent, the child's blood group will be
AB.
– If a child inherits the gene encoding hemoglobin A from either parent and
the gene encoding hemoglobin S from the other parent, the child's red
blood cells will contain roughly equal amounts of the two types of
hemoglobin.
But there are a few exceptions to this rule. A small number of genes in
mammals (~50 of them at the most recent count) have been found to
be imprinted. Because most imprinted genes are repressed, either
– the maternal (inherited from the mother) allele is expressed exclusively
because the paternal (inherited from the father) allele is imprinted or
– vice-versa.
74. Consistent correlation between gene
silencing (e.g. in B en T lymphocytes) and
presence in heterochromatin regions
– LCR, enhancers, insulators: act by maintaining
endogenous loci in a chromatin compartment
that is either transcr. permissive or
nonpermissive?
75. Position variegation
Position effects can be observed for the Drosophila white gene. Wild-type flies
with a normal white gene have red eyes. If the white gene is inactivated by
mutation, the eyes become white (hence the name of the gene). In flies with a
chromosomal inversion that moves the white gene near a heterochromatic
region, the eyes are mottled, with red and white patches. The white patches
represent cells where the white gene is silenced and red patches represent cells
that express the white gene. (After L.L. Sandell and V.A. Zakian, Trends Cell Biol.
2:10-14, 1992.)
76. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Alteration of chromatin structure during
transcription
Oncogenes
Techniques
77. The development and metastasis of human
colorectal cancer and its genetic basis.
A mutation in the APC tumor-suppressor gene in
a single epithelial cell causes the cell to divide,
although surrounding cells do not, forming a
mass of localized benign tumor cells called a
polyp. Subsequent mutations leading to
expression of a constitutively active Ras protein
and loss of two tumor-suppressor genes, DCC
and p53, generates a malignant cell carrying all
four mutations; this cell continues to divide and
the progeny invade the basal lamina that
surrounds the tissue. Some tumor cells spread
into blood vessels that will distribute them to
other sites in the body. Additional mutations
cause exit of the tumor cells from the blood
vessels and growth at distant sites; a patient with
such a tumor is said to have cancer. [Adapted
from B. Vogelstein and K. Kinzler, 1993, Trends
Genet. 9:101.]
78. Overview
Gene expression
Initiation of transcription
Regulation of transcription
Alteration of chromatin structure during
transcription
Oncogenes
Techniques
82. Sources
B Lewin, Genes VII
Lodish et al. Molecular Cell Biology
EH Davidson: Genomic Regulatory
Systems
Alberts et al. Essential Cell Biology
EM Blackwood & JT Kadonaga: Going the
distance: a current view of enhancer action
Cell, February 22, 2002: 108 (4) "Reviews
on Gene Expression"
Editor's Notes
RNA pol I: rRNA RNA pol II: mRNA RNA pol III: tRNA e.a. small RNAs
Transcription-control regions Signals for 3’ cleavage and polyadenylation Signals for splicing of primary RNA transcripts Mutations in these signals prevent expression of a functional mRNA and thus of the encoded protein
Different Sigma factors
Activator proteins communicate with the basal transcription machinery at promoters through intermediary factors. Two candidates for such factors have been identified, the TAF complex which interacts with TBP, and the Mediator complex which interacts with the RNA polymerase II CTD. TAFs enable a response to activators in a partially reconstituted Drosophila or human transcription system, while Mediator supports activation in a fully defined yeast system. Deletion or destruction of TAFs has, however, no effect upon induction or transcription of most genes in yeast in vivo. Inactivation of Mediator components, on the other hand, abolishes both induction of specific genes and transcription in general. It therefore appears that Mediator is the primary conduit of information from enhancers to promoters in vivo. A small number of yeast promoters do require TAFs for transcription in vivo, including those for G1 cyclins and for some cell cycle-independent genes. Dissection of these promoters identifies sequences surrounding the TATA box and not upstream activating elements as responsible for the TAF requirement. TAFs are evidently involved in promoter selection and specificity rather than enhancer-promoter interaction.
GAL80 prevents GAL4 from activating transcription: GAL80 is released when galactose is present, thus allowing GAL4 to activate its target genes
Repressors cancel the output of the activators
Drosophila, rho = Cell surface component, Signaling Neuroectodermal territory->ventral CNS Repression by Snail in the mesoderm mutated snail sites -> no more repression new snail sites somewhere else -> OK autonomous: 2 modules linked (rho element + eve stripe 2: Kr repressor, Bcd activator) -> A/P rho pattern + D/V eve stripe 2 2 x represson element of zen (with Dl sites) gene placed distal to the eve stripe 2 Deadringer & Cut corepressors interact at I
A1. Initial: vegetal plate of blastula-stage embryo -> endo+mesodermal cell types (but no skeletogenic)