Eukaryotic Gene Regulation
Page 1
Transcriptional Regulation in Eukaryotes
Many features of transcription and its regulation are similar to those in prokaryotes:
Genes are controlled at the level of transcription
Mechanisms of transcriptional regulation are similar to those found in bacteria
Trans-acting regulatory proteins & cis-acting regulatory target sequences
Prokaryotes Vs Eukaryotes
$1$ RNA Pol vs $3$ RNA Pol
Extensive processing of RNA transcripts
RNA polymerase II is much larger and more complex than its prokaryotic counterpart
Page 2
Regulation must be able to:
Turn off the expression of most genes in the genome (transcriptional silencing)
Generate thousands of patterns of gene expression with a limited number of regulatory proteins, how?
Basal transcription → activated transcription
Combinatorial interaction (modularity and cooperativity)
The regulatory regions of many eukaryotic genes are often longer than the gene itself
Page 3
Cis-acting regulatory elements: For RNA polymerase II to transcribe DNA into RNA at maximum speed, multiple cis-acting regulatory elements must act cooperatively.
3 classes, of elements on the basis of their relative locations:
1) The promoter
2) Promoter-proximal elements (the promoter) cis-acting sequences that bind to proteins that in turn assist the binding of RNA polymerase II to its promoter
3) Additional cis-acting elements independent of distance and position (enhancers and silencers)Often, an enhancer or silencer element acts in only one or a few cell types in a multicellular eukaryote
Page 4
The Promoter and Promoter-proximal Elements
Upstream promoter elements (UPEs) are found within $100$--$200$ bp of the transcription initiation site
Transcription factors that bind to promoter-proximal elements are constitutively expressed in all cells at all times, enabling activation of transcription in all cell types
Mutations in UPEs can have a dramatic effect on transcription
Page 5
Distance-Independent Cis-acting Elements
Eukaryotic genes are typically expressed at high levels in only a subset of tissues or in response to a signal (e.g., a hormone or a pathogen)
Enhancers can greatly increase transcription rates from promoters on the same DNA molecule (cis); activate or positively regulate transcription
Silencers are sequences bound by repressors, thereby inhibiting activators and reducing transcription
Enhancers and silencers are organized as a series of sequences
Act from a distance of $50$ kb upstream or downstream from the promoter
Page 6
Chromatin Structure and Gene Regulation
In the nucleus, histone proteins associate to form octamers around which helical DNA tightly coils to create chromatin
Transcription factors, activators, and RNA polymerase must bind to the DNA to initiate transcription
The ability of DNase I to digest DNA provides an indication of the DNA–histone association
Regions $1000$ nucleotides upstream from the start site of transcriptionally active genes become highly sensitive to the action of DNase I
Page 7
Chromatin organization (simplified level)
DNA is a double-stranded helical structure (2 nm per diameter)
DNA is complexed with histones to form nucleosomes
Each nucleosome consists of eight histone proteins around which the DNA wraps $1.65$ times
Histone H1
Nucleosomes form "beads" on DNA "string"; nucleosome core of eight histone molecules
Nucleosomes form a 300-nm fiber
The 300-nm fiber is folded into a $30$-nm fiber
The $30$-nm fiber is organized into a $250$-nm-wide fiber
The 250-nm fiber folds to produce the chromatid of a chromosome (dia. ~$700$ nm)
Page 8
Factors affecting Chromatin Structure
Histone acetylation
Histone domains
A globular domain (associates with other histones & the DNA)
A positively charged tail domain (interacts with the negatively charged phosphate backbone of DNA)
Certain transcription factors and other proteins that regulate transcription either have acetyltransferase activity or recruit acetyltransferases to the DNA
Chromatin-remodeling complexes (CRCs) alter chromatin structure without acetylating histone proteins
Page 9
Acetylation of histones controls flowering in Arabidopsis (chromatin and gene regulation)
Chromatin FLC (flowering locus C) w/ mRNA SUME
Acetylated chromatin
FLC encodes a regulatory protein that represses flowering
Transcriptional activator protein
No flowering takes place
Step 1: Acetyl groups on histone proteins destabilize chromatin structure
Step 2: FLC encodes a regulator protein that represses flowering
Step 3: No flowering takes place
Page 10
Acetylation of histones controls flowering in Arabidopsis (continued)
Chromatin FLC w/ SUME
Acetylated chromatin
FLD (Flowering locus D) encodes a deacetylase enzyme
Step 4: Flowering locus D (FLD) encodes a deacetylase enzyme that removes acetyl groups
Repression of flowering
Transcription of FLC is reduced, enabling flowering
Page 11
Acetylation of histones controls flowering in Arabidopsis (continued)
Step 5: Deacetylation via FLD restores chromatin structure
Step 6: No transcription of FLC takes place
Step 7: Flowering is not suppressed and thus occurs
Page 12
Chromatin Remodeling
Alters chromatin structure without altering the chemical structure of histones directly
CRCs bind directly to specific DNA sites and reposition nucleosomes
SWI–SNF (SWItch/Sucrose Non-Fermentable)
Found in yeast, humans, Drosophila, and other organisms
Uses ATP hydrolysis to reposition nucleosomes
Two mechanisms:
Nucleosome sliding
Conformational changes in DNA, in nucleosomes, or both
CRCs are targets of transcriptional activators or repressors and sometimes work with histone acetyltransferases
Page 13
DNA Methylation
Cytosine methylation (in vertebrates and plants)
Heavily methylated DNA → repression of transcription; unmethylated DNA transcriptionally active
DNA methylation is most common on CpG dinucleotides
CpG islands are commonly found near transcription start sites (TSS)
Methylation attracts deacetylases
DNA methylation in CpG islands is associated with transcriptional repression (repetitive sequences, transposable elements)
Page 14
Transcriptional Control in Eukaryotic Cells
Transcriptional activator proteins stimulate transcription by facilitating assembly or action of the basal transcription apparatus at the core promoter
Activators may interact directly with the basal apparatus or indirectly via protein coactivators
Acetyltransferase activity is often involved in this process
Page 15
GAL4 transcriptional activator in yeast
UASG acts as an enhancer
GAL4 contains an acidic activation domain that helps to recruit TFIIB
Mechanism: stimulate transcription by enhancing the ability of TFIIB
Upstream activating sequence (UAS)
Page 16
Eukaryotic Repressors
Repressors bind to sequences in the regulatory promoter or to distant sequences called silencers (distance- and position-independent cis-acting elements)
Eukaryotic repressors do not directly block RNA polymerase (unlike prokaryotic repressors)
Repression can occur by:
Competing with activators for DNA binding
Preventing activators from contacting the basal transcription machinery (BTA)
Direct interference with the assembly of the basal transcription apparatus
Page 17
Enhancers and Insulators
Enhancers: position- and distance-independent cis-acting elements; most enhancers can stimulate any nearby promoter
Insulators (boundary elements): DNA sequences that block or insulate the effect of enhancers in a position-dependent manner
Specific proteins bind to insulators and contribute to their blocking activity
Insulators help limit the spread of changes in chromatin structure after transcription
Page 18
Coordinated gene regulation
Eukaryotic cells do not possess operons
Several eukaryotic genes can be activated by the same stimulus (e.g., heat-shock proteins—about $20$ different genes)
Genes that are co-ordinately expressed share regulatory sequences in their promoters or enhancers (response elements)
A single eukaryotic gene may be regulated by several different response elements
Page 19
Continued: Coordinated gene regulation
A single gene may be activated by several different response elements, found in both promoters and enhancers
Multiple response elements allow the same gene to be activated by different stimuli
Same response element in different genes allows a single stimulus to activate multiple genes (e.g., Phorbol esters)
Page 20
Gene Control Through Messenger RNA Processing
Example: T-antigen gene of the mammalian virus SV40
Splicing factor 2 (SF2) enhances the production of mRNA encoding the small t antigen
Page 21
Sex differentiation in Drosophila
Sexual phenotype is determined by the X-to-autosome ratio (X:A ratio)
Key players: Sxl (sex-lethal), tra (transformer), dsx (doublesex)
Page 22
Sex differentiation in Drosophila (repeat for emphasis)
X:A ratio guides sex determination path via Sxl/tra/dsx network
Page 23
Sex differentiation in Drosophila (repeat for emphasis)
Sxl, tra, dsx operate in the regulatory cascade controlling female vs male development
Page 24
Sex differentiation in Drosophila (Sxl regulation of splicing)
Sxl protein (produced only in females) may block the upstream splice site on the tra pre-mRNA
This blockage forces the spliceosome to use the downstream 3' splice site
Result: production of Tra protein and ultimately female traits
Page 25
Gene Control Through RNA Stability
Eukaryotic mRNAs are generally more stable than bacterial mRNAs
mRNA stability ranges from minutes to months
Ribonucleases (about $10$ different types)
Several pathways of mRNA breakdown:
Removal of the 5' cap (5' to 3' direction)
Removal of the 3' end of the mRNA (3' to 5' direction)
Internal cleavage of the mRNA
Roles of the poly-A tail, PABPs
The 5' UTR, coding region, and the 3' UTR play regulatory roles in mRNA stability and translation
Page 26
RNA Interference (RNAi)
Posttranscriptional regulation of gene expression
Large double-stranded RNA (dsRNA) triggers RNAi
Pathways involve Dicer processing:
A large dsRNA is diced into small, double-stranded interfering RNAs (21–28 base pairs long)
Double-stranded interfering RNA forms double-stranded RNA segments that are loaded into RNA-induced silencing complex (RISC)
RISC uses single-stranded interfering RNA to target complementary mRNA
If perfectly base-paired, the mRNA is cleaved and degraded
If imperfectly base-paired, translation is repressed
sRNA categories: siRNA and miRNA
Page 27
Continued: Action of siRNA and miRNA
Dicer-generated siRNAs and miRNAs guide RISC to target mRNA
siRNA: typically perfect complementarity → cleavage and degradation
miRNA: often imperfect complementarity → translation repression
Outcomes: mRNA cleavage or translational repression, depending on pairing
Page 28
Sources of siRNA and miRNA
microRNA genes (mir) in eukaryotes
No protein-coding activity
Mutations affecting regulation of other genes
Computational analyses identify candidate mir genes
miRNA-targeted mRNA analyses focus on transcription factors or developmentally significant proteins
Transcriptions of transposons and transgenes may stimulate siRNA synthesis
RNA-dependent RNA polymerase (RdRP) in plants and nematodes relates to gene regulation or immunity against viruses
Page 29
Action of siRNA and miRNA (detailed pathway)
Step 1: A large double-stranded RNA is diced by Dicer into siRNAs/miRNAs
Step 2: siRNA/miRNA associates with RISC
Step 3: The siRNA/miRNA guides RISC to complementary mRNA sequences
Step 4: If perfect pairing, RISC cleaves the mRNA
Step 5: Cleaved mRNA is degraded
Step 6: If imperfect pairing, translation is inhibited
Step 7: Additional dsRNA regions may be processed into other siRNAs/miRNAs
Page 30
Action of siRNA and miRNA (continued)
RITS and related complexes can target DNA methylation or histone modifications to reinforce transcriptional silencing
Methylating enzymes may methylate DNA or histones to inhibit transcription
Page 31
Epigenetic Inheritance: DNA Methylation
A heritable trait mappable to a locus, but not involving a change in the DNA sequence at that locus
About $2$–$7\ imes 10^{-2}$% of $40\%$ G:C is methylated
HpaII recognizes and cleaves the sequence $C ext{C}G ext{G}$ (with methylation status affecting cleavage)
Methylated and unmethylated DNAs give different patterns of restriction fragments when digested with HpaII
CpG dinucleotides occur less often than expected in mammalian genomes; CpG→TpG due to methylation
Methylation of CpG islands is associated with transcriptional repression (repetitive sequences, transposable elements)
CpG islands (1–2 kb): ~30,000 in the human genome, often near TSS
DNAse I hypersensitivity & MeCP2 mutation implications
De novo methylation vs maintenance methylation
The “methylome” reflects heritable changes in methylation patterns
Page 32
Imprinting and parental origin of expression
Control of gene expression by parental origin (imprinting)
In mice, the Igf2 gene is expressed when inherited from the father and silenced when inherited from the mother
Methylation of CpG dinucleotides near Igf2 is established in the parental germ line
~20 imprinting sites in mice or humans
Page 33
Imprinting mechanism (Igf2 example)
Alleles of the Igf2 gene are imprinted in parental germ lines: methylated in the female germ line and unmethylated in the male germ line
Imprinted alleles from each parent are combined in the zygote at fertilization
During somatic development, the maternally contributed allele remains methylated while the paternally contributed allele remains unmethylated; only the unmethylated (paternally contributed) allele is expressed in somatic cells
During development of the germ line, the methylation imprint is erased
Page 34
Oogenesis vs Spermatogenesis in Igf2 imprinting
Oogenesis: Igf2 gene becomes methylated in the maternal germ line
Spermatogenesis: Igf2 gene remains unmethylated in the paternal germ line
If the mouse is female, all Igf2 genes will be methylated (inherited from mother) despite paternal copies
If the mouse is male, none of the Igf2 genes are methylated despite maternal copies
Result: imprinting patterns are sex-specific and reset each generation
Page 35
Summary of imprinting concepts
The methylation imprint is established in the parental germ line
A methylated gene inherited from one sex can be unmethylated in offspring of the opposite sex
Imprints are reset each generation depending on the sex of the animal
Some genes are methylated in one sex but not the other, implying sex-specific factors control the methylation machinery
Page 36
X-chromosome Inactivation
X-inactivation center (XIC)
X inactive specific transcript (XIST) acts in cis
XIST is ~17 kb, non-coding, not an ORF, poly-A, and remains in the nucleus to coat the entire X chromosome
Random X-inactivation occurs at ~1000-cell level
Steps often described as: counting, choice, inactivation
Page 37
Chromosome Numbers in Different Species
Common name vs Genus and Species; Diploid chromosome numbers:
Buffalo: 60
Cat: 38
Cattle: 60
Dog: 78
Donkey: 62
Goat: 60
Horse: 64
Human: 46
Pig: 38
Sheep: 54
Page 38
The Extremes in Chromosome Number
Minimum: Ants, Myrmecia pilosula; females have a single pair of chromosomes (haplodiploidy: fertilized eggs become females, unfertilized eggs become males)
Maximum: Ferns, Ophioglossum reticulatum; ~630 pairs of chromosomes (1260 chromosomes per cell)
Despite enormous chromosome numbers, cells can accurately segregate during mitosis
Page 39
Genome Sequencing and Exome
Exome: All of the axons? protein-coding sequences; about $1\%$ of the genome; cost around $
$2300 USDWhat’s in the genome?
22,500 protein-coding genes (plus splice variants)
~50% are enzymes; rest are structural proteins
503 miRNA-encoding genes (in mice)
Many non-coding RNA genes (~8x more than protein-coding genes)
Non-coding RNA genes include clues about regulation and development
Page 40
Human Genetics: Human Genome Project (HGP)
Key figures: Jim Watson and Craig Venter (first two people sequenced)
Genome size: ~3 billion base pairs (bp) → ~3×10^9 bp
SNPs: ~3×10^6 SNPs differentiating individuals; ~0.1% of genome differences between any two individuals
In protein-coding portions: ~14,779 SNPs; ~20% are private SNPs (not seen in other person)
2020-change in protein sequence: non-synonymous substitutions; ~12% predicted to disrupt protein function; ~11% in genes with clear disease associations (recessive mutations)
Mutation rate: ≈ $1.1×10^{-8}$ bp per generation
Page 41
Human Genome Project (re-cap)
Summary of SNPs and mutation rates reaffirming that genetic variation underpins human diversity and disease associations
Emphasizes the ongoing importance of genome sequencing and interpretation for understanding health, disease, and evolution
Page 1 - Transcriptional Regulation in Eukaryotes
Many features of transcription and its regulation in eukaryotes are similar to those in prokaryotes. This includes genes being controlled at the level of transcription, and the mechanisms of transcriptional regulation resembling those found in bacteria, involving trans-acting regulatory proteins and cis-acting regulatory target sequences. However, key differences exist: eukaryotes have RNA Polymerases compared to prokaryotes' RNA Pol, and there is extensive processing of RNA transcripts. RNA polymerase II, in particular, is significantly larger and more complex than its prokaryotic counterpart.
Page 2 - Regulation must be able to:
Transcriptional regulation in eukaryotes must be capable of several critical functions. It needs to turn off the expression of most genes in the genome, a process known as transcriptional silencing. Furthermore, it must generate thousands of diverse patterns of gene expression with a limited number of regulatory proteins. This is achieved through the transition from basal transcription to activated transcription, relying on combinatorial interaction, which involves modularity and cooperativity. It is notable that the regulatory regions of many eukaryotic genes are often longer than the gene itself.
Page 3 - Cis-acting regulatory elements:
For RNA polymerase II to transcribe DNA into RNA at its maximum speed, multiple cis-acting regulatory elements must cooperatively interact. These elements are categorized into three classes based on their relative locations:
The promoter: This is a fundamental cis-acting element.
Promoter-proximal elements: These are cis-acting sequences located near the promoter that bind to proteins, which in turn assist the binding of RNA polymerase II to its promoter.
Additional cis-acting elements independent of distance and position: These include enhancers and silencers. Often, an enhancer or silencer element acts in only one or a few specific cell types within a multicellular eukaryote.
Page 4 - The Promoter and Promoter-proximal Elements
Upstream promoter elements (UPEs) are located within - bp of the transcription initiation site. Transcription factors that bind to these promoter-proximal elements are constitutively expressed in all cells at all times, thereby enabling the activation of transcription across all cell types. Mutations within UPEs can significantly impact transcription levels.
Page 5 - Distance-Independent Cis-acting Elements
Eukaryotic genes are typically expressed at high levels only in a subset of tissues or in response to specific signals like hormones or pathogens. Enhancers are cis-acting elements that can drastically increase transcription rates from promoters located on the same DNA molecule, thus acting as positive regulators of transcription. Conversely, silencers are sequences bound by repressor proteins, which inhibit activators and consequently reduce transcription. Both enhancers and silencers are organized as a series of distinct sequences and can exert their effects from a distance of up to kb, whether upstream or downstream from the promoter.
Page 6 - Chromatin Structure and Gene Regulation
Inside the nucleus, histone proteins aggregate to form octamers around which helical DNA tightly coils, forming chromatin. For transcription to initiate, transcription factors, activators, and RNA polymerase must bind to the DNA. The susceptibility of DNA to digestion by DNase I indicates the strength of the DNA–histone association. Regions approximately nucleotides upstream from the transcription start site in active genes become highly sensitive to DNase I action, indicating an open chromatin structure.
Page 7 - Chromatin organization (simplified level)
At a simplified level of chromatin organization, DNA itself is a double-stranded helical structure with a diameter of nm. This DNA is complexed with histones to form nucleosomes. Each nucleosome consists of eight histone proteins around which the DNA wraps approximately times. Histone H1 then aids in compacting these nucleosomes, which appear as 'beads' on a DNA 'string', into a higher-order structure. These nucleosomes subsequently form a -nm fiber, which is further folded into a -nm fiber. The -nm fiber is then organized into a -nm-wide fiber, which ultimately folds to produce the chromatid of a chromosome, measuring approximately nm in diameter.
Page 8 - Factors affecting Chromatin Structure
Several factors can affect chromatin structure, including histone acetylation and chromatin-remodeling complexes (CRCs). Histone proteins possess a globular domain that associates with other histones and DNA, and a positively charged tail domain that interacts with the negatively charged phosphate backbone of DNA. Certain transcription factors and other regulatory proteins can either possess acetyltransferase activity themselves or recruit acetyltransferases to the DNA, leading to histone acetylation. Additionally, chromatin-remodeling complexes (CRCs) alter chromatin structure without directly acetylating histone proteins.
Page 9 - Acetylation of histones controls flowering in Arabidopsis (chromatin and gene regulation)
In Arabidopsis, the acetylation of histones plays a crucial role in controlling flowering, specifically through the FLC (Flowering Locus C) gene. The FLC gene in chromatin, along with mRNA SUME, is involved. Acetylated chromatin is a key state. FLC encodes a regulatory protein that represses flowering. This regulatory protein acts as a transcriptional activator, leading to no flowering taking place. The process initiated by acetyl groups on histone proteins destabilizes chromatin structure. Consequently, FLC encodes its repressor protein, and flowering is suppressed.
Page 10 - Acetylation of histones controls flowering in Arabidopsis (continued)
The regulation of flowering in Arabidopsis continues with Chromatin FLC and SUME, interacting within acetylated chromatin. The FLD (Flowering Locus D) gene encodes a deacetylase enzyme. This enzyme removes acetyl groups from histones, which consequently reduces the repression of flowering. As a result, the transcription of FLC is reduced, thereby enabling flowering to occur.
Page 11 - Acetylation of histones controls flowering in Arabidopsis (continued)
The final steps in the control of flowering in Arabidopsis by histone acetylation involve deacetylation via FLD. This enzyme restores the chromatin structure. With the chromatin structure restored, no transcription of FLC takes place. Consequently, flowering is not suppressed and thus occurs.
Page 12 - Chromatin Remodeling
Chromatin remodeling alters chromatin structure without directly changing the chemical structure of histones. Chromatin-remodeling complexes (CRCs) bind directly to specific DNA sites and reposition nucleosomes. A well-known example is SWI–SNF (SWItch/Sucrose Non-Fermentable), found in yeast, humans, Drosophila, and other organisms. These complexes utilize ATP hydrolysis to reposition nucleosomes through two main mechanisms: nucleosome sliding or conformational changes in DNA, nucleosomes, or both. CRCs are often targets of transcriptional activators or repressors and sometimes work in conjunction with histone acetyltransferases.
Page 13 - DNA Methylation
Cytosine methylation is a significant form of gene regulation observed in vertebrates and plants. Heavily methylated DNA is typically associated with the repression of transcription, while unmethylated DNA remains transcriptionally active. DNA methylation most commonly occurs on CpG dinucleotides. CpG islands, which are regions rich in CpG dinucleotides, are frequently found near transcription start sites (TSS). Methylation at these sites can attract deacetylases, contributing to transcriptional repression. This form of methylation in CpG islands is also associated with the silencing of repetitive sequences and transposable elements.
Page 14 - Transcriptional Control in Eukaryotic Cells
Transcriptional activator proteins in eukaryotic cells stimulate transcription by promoting the assembly or action of the basal transcription apparatus at the core promoter. These activators may interact directly with the basal apparatus or indirectly through protein coactivators. Acetyltransferase activity is frequently involved in this process, facilitating changes in chromatin structure that favor transcription.
Page 15 - GAL4 transcriptional activator in yeast
The GAL4 transcriptional activator in yeast provides a notable example of transcriptional control. Its Upstream Activating Sequence (UASG) acts as an enhancer. The GAL4 protein contains an acidic activation domain that aids in recruiting TFIIB. The mechanism by which GAL4 stimulates transcription involves enhancing the ability of TFIIB to function within the basal transcription machinery, thereby leading to increased gene expression.
Page 16 - Eukaryotic Repressors
Eukaryotic repressors function by binding to specific sequences located in the regulatory promoter or to distant sequences known as silencers, which are considered distance- and position-independent cis-acting elements. Unlike prokaryotic repressors, eukaryotic repressors do not directly block RNA polymerase. Instead, repression can occur through several mechanisms: repressors may compete with activators for DNA binding sites, prevent activators from contacting the basal transcription machinery (BTA), or directly interfere with the assembly of the basal transcription apparatus.
Page 17 - Enhancers and Insulators
Enhancers are position- and distance-independent cis-acting elements that can stimulate any nearby promoter. In contrast, insulators, also known as boundary elements, are DNA sequences that block or insulate the effect of enhancers in a position-dependent manner. Specific proteins bind to insulators and contribute to their blocking activity, helping to limit the spread of changes in chromatin structure after transcription.
Page 18 - Coordinated gene regulation
Eukaryotic cells do not possess operons, yet they can achieve coordinated gene regulation. Several eukaryotic genes can be activated by the same stimulus, such as the approximately different heat-shock protein genes. Genes that are coordinately expressed typically share common regulatory sequences in their promoters or enhancers, which are known as response elements. Importantly, a single eukaryotic gene can be regulated by several different response elements, allowing for complex control.
Page 19 - Continued: Coordinated gene regulation
Coordinated gene regulation in eukaryotes highlights that a single gene may be activated by several different response elements, found in both promoters and enhancers. This multiplicity of response elements enables the same gene to be activated by various stimuli. Conversely, the presence of the same response element in different genes allows a single stimulus, such as Phorbol esters, to activate multiple genes, demonstrating a sophisticated network of gene control.
Page 20 - Gene Control Through Messenger RNA Processing
Gene control can also occur through messenger RNA (mRNA) processing. An illustrative example of this is the T-antigen gene of the mammalian virus SV40. In this system, the splicing factor 2 (SF2) plays a crucial role by enhancing the production of mRNA that encodes the small t antigen, demonstrating how alternative splicing can regulate gene expression.
Page 21 - Sex differentiation in Drosophila
Sex differentiation in Drosophila is determined by the X-to-autosome ratio (X:A ratio). Key players in this process are the Sxl (sex-lethal), tra (transformer), and dsx (doublesex) genes, which form a regulatory cascade.
Page 22 - Sex differentiation in Drosophila (repeat for emphasis)
The X:A ratio guides the sex determination pathway in Drosophila through the intricate Sxl/tra/dsx gene network, emphasizing its central role in establishing sexual phenotype.
Page 23 - Sex differentiation in Drosophila (repeat for emphasis)
Sxl, tra, and dsx genes are integral components that operate within the regulatory cascade, meticulously controlling the development of either female or male characteristics in Drosophila.
Page 24 - Sex differentiation in Drosophila (Sxl regulation of splicing)
In the context of sex differentiation in Drosophila, the Sxl protein, which is produced exclusively in females, can block the upstream splice site on the tra pre-mRNA. This blockage forces the spliceosome to use the downstream 3' splice site. The consequence of this alternative splicing is the production of the Tra protein, which ultimately leads to the development of female traits.
Page 25 - Gene Control Through RNA Stability
Gene control can also be exerted through RNA stability. Eukaryotic mRNAs are generally more stable than bacterial mRNAs, with their stability ranging from minutes to months. mRNA breakdown is facilitated by various ribonucleases, of which there are approximately different types. Several pathways contribute to mRNA degradation, including the removal of the 5' cap (proceeding in the 5' to 3' direction), the removal of the 3' end of the mRNA (proceeding in the 3' to 5' direction), and internal cleavage of the mRNA. The poly-A tail and poly-A-binding proteins (PABPs) play significant roles in these processes. Furthermore, the 5' UTR, coding region, and 3' UTR of the mRNA also contribute regulatory roles in both mRNA stability and translation.
Page 26 - RNA Interference (RNAi)
RNA Interference (RNAi) is a crucial posttranscriptional regulatory mechanism of gene expression, triggered by large double-stranded RNA (dsRNA). The pathways involved in RNAi utilize Dicer processing. Initially, a large dsRNA is diced into small, double-stranded interfering RNAs, typically - base pairs long. These double-stranded interfering RNAs then form double-stranded RNA segments that are loaded into the RNA-induced silencing complex (RISC). RISC subsequently uses a single-stranded interfering RNA to target complementary mRNA. If there is perfect base-pairing, the mRNA is cleaved and degraded. If the pairing is imperfect, translation is repressed instead. Small RNA (sRNA) categories involved in RNAi include siRNA and miRNA.
Page 27 - Continued: Action of siRNA and miRNA
The action of siRNA and miRNA continues to guide RISC to target mRNA after being generated by Dicer. Specifically, siRNAs typically exhibit perfect complementarity with their target mRNA, leading to cleavage and degradation of the mRNA. In contrast, miRNAs often show imperfect complementarity, which primarily results in the repression of translation. Therefore, the outcomes of RISC activity, whether mRNA cleavage or translational repression, depend on the degree of base pairing between the sRNA and the target mRNA.
Page 28 - Sources of siRNA and miRNA
MicroRNA genes (mir) are sources of miRNA in eukaryotes; these genes do not have protein-coding activity and mutations affecting them can impact the regulation of other genes. Computational analyses are used to identify candidate mir genes, and studies of miRNA-targeted mRNAs often focus on transcription factors or developmentally significant proteins. Transcription of transposons and transgenes can also stimulate siRNA synthesis. Additionally, RNA-dependent RNA polymerase (RdRP) in plants and nematodes is relevant to gene regulation or immunity against viruses.
Page 29 - Action of siRNA and miRNA (detailed pathway)
The detailed pathway for the action of siRNA and miRNA begins with a large double-stranded RNA being diced by Dicer into siRNAs/miRNAs. Subsequently, the siRNA/miRNA associates with RISC. This complex then guides RISC to complementary mRNA sequences. If perfect pairing occurs, RISC cleaves the mRNA, which is then degraded. However, if imperfect pairing is observed, translation is inhibited. It is also possible for additional dsRNA regions to be processed into other siRNAs/miRNAs.
Page 30 - Action of siRNA and miRNA (continued)
Beyond direct mRNA cleavage or translational repression, the action of siRNA and miRNA can extend to influencing chromatin. RITS and related complexes are capable of targeting DNA methylation or histone modifications. This action helps to reinforce transcriptional silencing. Methylating enzymes, often recruited by these complexes, can methylate DNA or histones, further inhibiting transcription and contributing to long-term gene silencing.
Page 31 - Epigenetic Inheritance: DNA Methylation
Epigenetic inheritance refers to a heritable trait that can be mapped to a locus but does not involve a change in the underlying DNA sequence at that locus. In mammalian genomes, approximately -% of the % G:C content is methylated. The restriction enzyme HpaII recognizes and cleaves the sequence , and its cleavage efficiency is affected by methylation status, allowing for differentiation between methylated and unmethylated DNAs based on restriction fragment patterns. CpG dinucleotides occur less often than expected in mammalian genomes, primarily due to methylation-induced deamination of cytosine to thymine (). Methylation of CpG islands, which are - kb regions commonly found near transcription start sites (about in the human genome), is strongly associated with transcriptional repression, particularly for repetitive sequences and transposable elements. The implications of DNase I hypersensitivity and MeCP2 mutations also highlight the role of chromatin accessibility and methylation in gene regulation. DNA methylation can occur as de novo methylation (new methylation events) or maintenance methylation (copying existing methylation patterns during replication). The concept of the “methylome” encompasses these heritable changes in methylation patterns.
Page 32 - Imprinting and parental origin of expression
Imprinting refers to the control of gene expression based on parental origin. A classic example is the Igf2 gene in mice: it is expressed when inherited from the father but silenced when inherited from the mother. This differential expression is established by the methylation of CpG dinucleotides near the Igf2 gene in the parental germ line. There are approximately known imprinting sites in mice and humans, demonstrating that this mechanism is not unique to Igf2.
Page 33 - Imprinting mechanism (Igf2 example)
The imprinting mechanism for the Igf2 gene involves differential methylation established in the parental germ lines: it is methylated in the female germ line and unmethylated in the male germ line. These imprinted alleles from each parent are combined in the zygote at fertilization. During subsequent somatic development, the maternally contributed allele remains methylated, while the paternally contributed allele remains unmethylated. Consequently, only the unmethylated (paternally contributed) allele is expressed in somatic cells. However, during the development of the germ line in the offspring, the methylation imprint is erased, allowing for sex-specific re-establishment of the imprint in the next generation.
Page 34 - Oogenesis vs Spermatogenesis in Igf2 imprinting
In Igf2 imprinting, there are distinct patterns during oogenesis and spermatogenesis. During oogenesis, the Igf2 gene becomes methylated in the maternal germ line. Conversely, during spermatogenesis, the Igf2 gene remains unmethylated in the paternal germ line. This means that if a mouse is female, all Igf2 genes will be methylated, regardless of whether they were originally paternal copies. If a mouse is male, none of the Igf2 genes will be methylated, despite having maternal copies. As a result, imprinting patterns are sex-specific and are reset in each generation to reflect the sex of the individual producing the gametes.
Page 35 - Summary of imprinting concepts
To summarize imprinting concepts, the methylation imprint is fundamentally established in the parental germ line. A key characteristic is that a methylated gene inherited from one sex can become unmethylated in offspring of the opposite sex. Furthermore, imprints are reset in each generation depending on the sex of the animal. This dynamic control suggests that some genes are methylated in one sex but not the other, implying that sex-specific factors control the methylation machinery responsible for these epigenetic marks.
Page 36 - X-chromosome Inactivation
X-chromosome inactivation is a crucial dosage compensation mechanism in mammals. It is governed by the X-inactivation center (XIC). Within the XIC, the X inactive specific transcript (XIST) plays a central role. XIST is a long non-coding RNA, approximately kb in length, lacking an open reading frame (ORF), and it is poly-adenylated. Importantly, XIST remains in the nucleus and coats the entire X chromosome in cis. This random X-inactivation process typically occurs at the approximately -cell stage during embryonic development. The steps involved are often described as counting, choice, and inactivation.
Page 37 - Chromosome Numbers in Different Species
Here is a list of diploid chromosome numbers for various common species:
Buffalo:
Cat:
Cattle:
Dog:
Donkey:
Goat:
Horse:
Human:
Pig:
Sheep:
Page 38 - The Extremes in Chromosome Number
Chromosome numbers exhibit remarkable variation across species, ranging from very few to extraordinarily many. At the minimum end, the ant Myrmecia pilosula presents an extreme case where females possess a single pair of chromosomes, illustrating haplodiploidy where fertilized eggs develop into females and unfertilized eggs into males. Conversely, the fern Ophioglossum reticulatum holds the record for the maximum number, with approximately pairs of chromosomes, totaling chromosomes per cell. Despite these enormous chromosome numbers, cells in such organisms maintain the ability to accurately segregate them during mitosis.
Page 39 - Genome Sequencing and Exome
Genome sequencing aims to determine the entire genetic makeup of an organism, while the exome specifically refers to all of the protein-coding sequences, comprising about % of the genome. Exome sequencing currently costs approximately USD. The human genome contains roughly protein-coding genes, along with various splice variants. Approximately % of these genes are enzymes, with the remainder being structural proteins. Beyond protein-coding genes, the genome also includes about miRNA-encoding genes (in mice) and many other non-coding RNA genes, numbering approximately times more than protein-coding genes. These non-coding RNA genes provide crucial clues about gene regulation and development.
Page 40 - Human Genetics: Human Genome Project (HGP)
The Human Genome Project (HGP) was a landmark scientific endeavor. Key figures associated with its early sequencing efforts include Jim Watson and Craig Venter, the first two individuals to have their genomes sequenced. The human genome size is approximately billion () base pairs (bp). Single Nucleotide Polymorphisms (SNPs) account for about differences between individuals, meaning approximately % of the genome differs between any two individuals. Within protein-coding regions, there are about SNPs, with roughly % being private SNPs (not observed in other sequenced individuals). Changes in protein sequence, or non-synonymous substitutions, were estimated in , with approximately % predicted to disrupt protein function and about % found in genes with clear disease associations (often recessive mutations). The human mutation rate is estimated to be approximately $$