Differential Gene Expression PDF (INTARMED 2030)

DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Each gives rise to different cell types with different TABLE OF CONTENTS functions...

DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Each gives rise to different cell types with different TABLE OF CONTENTS functions I. Differential Gene Expression A. Background a. Fertilization b. Differentiation B. Differential Gene Expression a. Genomic Equivalence b. Somatic Cell Nuclear Transfer c. Three Postulates of Differential Gene Expression d. Levels of Control of Gene Figure 1: Germ layer derivatives Expression C. Basic Structure of a Eukaryotic Gene DIFFERENTIATION _______________________________________________________ II. Mechanisms of Differentiation A. Transcription Differentiation ➔ Generation of specialized cell types, with a. Chromatin Structure and Forms specialized structures and functions b. Histone Acetylation Different cell types can be generated from cells with c. Histone Methylation the same DNA as other cells d. DNA Methylation ○ Reason: Different cells express different sets of e. Cis-acting Elements genes at different times f. Transcription Factors ○ Why different cells produce different types of B. RNA Processing proteins a. Cap Addition b. Poly-A Addition c. Alternative Splicing C. Translation a. Differential mRNA Longevity b. Selective Localization c. RNA Interference D. Post-Translational Regulation I. DIFFERENTIAL GENE EXPRESSION BACKGROUND FERTILIZATION _______________________________________________________ Figure 2: Differentiation Fertilization ➔ Fusion of sperm and egg (gametes) ➔ Produces a single cell (zygote) DIFFERENTIAL GENE EXPRESSION The zygote becomes a blastula (through mitosis), then a gastrula (through morphogenetic movements) Dividing cells of fertilized egg form three distinct embryonic germ layers: ○ Ectoderm (outer layer) ○ Mesoderm (middle layer) ○ Endoderm (internal layer) Figure 3: An example of differential gene expression BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 1 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Cells 1, 2, and 3 have the same genes (ABCDEFGH) ○ Retain potential for being expressed Each cell expresses a different set of genes Only a percentage of the genome is expressed in the Varying proteins (triangles) from differential gene cell, and a portion of the RNA synthesized in each cell expression give rise to different cell types is specific for that cell type DIFFERENT LEVELS OF CONTROL OF GENE EXPRESSION _______________________________________________________ Gene expression The control of gene expression occurs at many levels ➔ The process of transcription and translation Transcription (central dogma) ➔ As mRNA is synthesized from DNA Differential gene expression Enhancers/activators, silencers/repressors ➔ The process by which cells become different from Regulation of chromatin structure by histone one another as a result of different combinations modification of genes expressed in different cells DNA methylation and demethylation ○ RNA processing ➔ mRNA is further modified, usually through splicing ➔ After this level, mRNA is transported out of the nucleus ○ Translation ➔ Some mRNA are degraded/not translated Differential mRNA stability Selective inhibition and activation of mRNA translation Selective localization of mRNA RNA-induced gene silencing Figure 4: Central dogma of molecular biology Control of RNA translation by cytoplasmic localization GENOMIC EQUIVALENCE _______________________________________________________ ○ Post-translational control of protein activity DNA in zygote is the same as the one in the nuclei of specialized cells due to mitosis Genomic equivalence ➔ Each somatic cell nucleus has the same number of chromosomes and therefore the same set of genes as all other somatic nuclei SOMATIC CELL NUCLEAR TRANSFER _______________________________________________________ Evidence of genomic equivalence Hypothesizes that if each cell’s nucleus is identical to the zygote nucleus, it should be capable of directing the entire development of the organism when transplanted into an activated enucleated egg General Process ○ The nucleus from a differentiated udder cell was implanted into an enucleated egg (from a different strain of sheep) ○ A surrogate ewe carried the egg and bore Dolly, the first clone Proves that the nucleus of a somatic cell has all the information to generate all cells in the body THREE POSTULATES OF DIFFERENTIAL GENE EXPRESSION _______________________________________________________ Figure 5: Levels in which gene expression occurs The DNA of all differentiated cells are identical The unused genes in differentiated cells are neither destroyed nor mutated BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 2 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 BASIC STRUCTURE OF A EUKARYOTIC GENE _______________________________________________________ Figure 6: Structure of a eukaryotic gene Promoter Figure 7: Parts of chromatin ➔ Site where RNA polymerase binds to initiate transcription Chromatins can be categorized into two forms based Exon on degree of coiling ➔ Sequence that is transcribed and translated Heterochromatin Intron ➔ Consists of tightly packed chromatin ➔ Sequence that is transcribed but not translated ➔ Genes are not accessible to RNA polymerase and ➔ Removed during RNA processing transcription factors Poly-A signal ◆ RNA polymerase = enzyme responsible for ➔ Sequence where poly-A tail will be added to mRNA making RNA from DNA ➔ Makes mRNA stable and translation efficient ◆ Transcription factors = other proteins that Transcription termination region bind to DNA to aid transcription regulation ➔ Region where transcription ends ➔ Genes are not transcribed and inactive Euchromatin ➔ Consists of loosely packed chromatin II. Mechanisms of Differentiation ➔ Genes are accessible to RNA polymerase and transcription factors Control of gene expression occurs at different levels ➔ Genes are transcribed and active The packing of a chromatin region could be regulated by different mechanisms such as histone acetylation TRANSCRIPTION and methylation Transcription HISTONE ACETYLATION _______________________________________________________ ➔ Synthesis of mRNA from DNA Promotes transcription Mechanisms for regulation of gene expression in this If histones are acetylated, the chromatin becomes level: loosely packed and is transcribed ○ Histone acetylation and methylation ○ Histones have a lot of basic amino acids, such as ○ DNA methylation lysine ○ Transcription factors ○ Lysine residues (+) in the histones are acetylated (-) and the charges are neutralized CHROMATIN STRUCTURE AND FORMS _______________________________________________________ ○ Neutralized charges result in less tight packing of Nucleosome chromatin, making it euchromatic. ➔ Basic unit of chromatin Addition of acetyl group is done by an enzyme called ➔ Composed of histones histone acetyltransferase (HAT) Histones Reversible: Deacylating histones leads to tight packing ➔ 8 in a nucleosome of chromatin structure → not transcribed ◆ 2 H2A, 2 H2B, 2 H3, 2 H4 ○ With help of enzyme histone deacetylase (HDAC) ➔ Wrapped around by 147 base pairs of DNA ○ Chromatin becomes heterochromatic Linker DNA ➔ Part of the DNA found between nucleosomes ➔ Where H1 binds H1 ➔ Fifth histone ➔ Facilitates and stabilizes coiling of nucleosomes into a solenoid structure BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 3 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 DNA methylation pattern differs at different stages of development ○ Different genes for globin protein are transcribes at weeks 6 and 12 At 6 weeks: promoter of ε-globin gene is unmethylated; promoter of γ-globin gene is methylated Gene of ε-globin is transcribed Gene of γ-globin is not transcribed At 12 weeks: promoter of γ-globin gene is unmethylated while the promoter of ε-globin gene is methylated Figure 8: Histone acetylation and deacetylation Gene of γ-globin is transcribed Gene of ε-globin is not transcribed HISTONE METHYLATION _______________________________________________________ Prevents transcription Histones are methylated by histone methyltransferase The nucleosome is condensed and transcription is usually prevented Promotion or inhibition of transcription depends on (1) the amino acid being methylated and (2) the presence of other methyl or acetyl groups Figure 11: DNA methylation of globin protein at 6 and 12 weeks CIS-ACTING ELEMENTS _______________________________________________________ Cis-acting Elements ➔ DNA sequence that control gene expression on Figure 9: Role of methylated amino acids in promotion or the same chromosome inhibition of transcription ➔ Binds proteins to regulate transcription ➔ Ex: Promoters, proximal/distal control elements DNA METHYLATION Proximal Control Elements _______________________________________________________ ➔ Near the promoter and gene it controls Inhibits transcription Distal Control Elements DNA itself could be methylated especially in cytosine ➔ Hundreds or thousands of nucleotides away from residues that are followed by guanine the gene they regulate C-G portions of DNA are magnets for methylation ➔ Could be enhancers or silencers Methylated DNA blocks binding of transcription factors Enhancers ➔ Increase rate of transcription ➔ Not necessary for transcription to occur Silencers ➔ Inhibit transcription Figure 10: Methyl group blocks Egr1 transcription factor Figure 12: Cis-acting elements in an mRNA and inhibits transcription Methylated DNA can recruit proteins that initiate histone methylation or deacetylation BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 4 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 TRANSCRIPTION FACTORS Activators: bind to enhancers _______________________________________________________ Repressors: bind to silencers Transcription Factors (TFs) Not required for binding of RNA polymerase ➔ Other proteins that bind to the DNA sequences to the promoter like the cis-acting elements Also called as trans-acting elements as they ➔ Influence ability of RNA polymerase to transcribe a could be encoded by genes in a different given gene chromosome TFs are required for the initiation of transcription ○ Indicate where transcription should start, in TFs are responsible for differential gene expression as eukaryotes the specific set of TFs in a cell determines which genes ○ Ex: A TF called TFIID binds to the TATA box, found are expressed in the promoter. Then, other TFs would also bind, ○ All cells contain the same DNA, silencers, and followed by RNA polymerase which would help enhancers due to genomic equivalence but not all initiate transcription cell contain the same set of TFs ○ Ex: Both liver and lens cells contain the same genes and enhancers for albumin and crystallin genes In the liver cell, only the albumin gene is expressed since the general TF that promotes transcription of albumin gene is present (for crystallin - absent) In lens cell, only the crystallin gene is expressed since the general TF that promotes transcription of crystallin gene is present (for albumin - absent) Figure 13: Role of transcription factors in initiation of transcription Figure 14: Transcription factors of albumin gene and There are two types of TFs: General TFs and Regulatory crystallin gene in liver cell nucleus and lens cell nucleus TFs ○ General TFs are required for binding of RNA TFs also serve as a connection between distal polymerase to the promoter cis-acting elements and the gene it regulates as the Ex: Proteins TFIID, B, F, E, H in Figure 13 DNA loops responsible for normal or basal level of transcription ○ Regulatory TFs control the rate of the basal level RNA PROCESSING of transcription Increase or decrease the rate of transcription RNA Processing Binds to the silencers or enhancers ➔ Involves modifications to mRNA before it Could be categorized as activators or undergoes translation repressors BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 5 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Primary RNA Transcript / nRNA / pre-mRNA ➔ RNA transcribed directly from DNA ➔ Undergoes processing before being translated ➔ In this stage, poly-A signal and introns are still transcribed Figure 16: Alternative splicing There are proteins that recognize exons and put them together, tasked by cell signaling. TRANSLATION Figure 15: Modifications in pre-mRNA Translation CAP ADDITION ➔ Converting mRNA into protein sequence _______________________________________________________ ➔ Controls gene expression by deciding on the fate A cap is added at the 5’ end (Figure 15), the side that is of mRNA (e.g. viability for degradation of mRNA, synthesized first, after a few nucleotides have been longevity of mRNA duration in cytoplasm, etc.) added. Cap structure: phosphate group attached to the 5’ end DIFFERENTIAL MRNA LONGEVITY _______________________________________________________ with 7-methylguanosine (G) The stability of mRNA can regulate translation Attaches when the transcript is 20-25 nucleotides long ○ More stable / longer lifespan of mRNA → greater ○ Attaches even before transcription is completed chance for it to be translated Cap is significant to the binding of mRNA to ribosome ○ If mRNA persists longer in cytoplasm, it can produce more proteins POLY-A TAIL ADDITION _______________________________________________________ ○ Stability is dependent on the length of poly-A tail Poly-A signal The length of poly-A tail is being controlled by 3’ ➔ Present in DNA and transcribed in mRNA (Figure untranslated region (UTR) 15) 3’ Untranslated Region (UTR) ➔ Where RNA is cleaved and poly-A is added ➔ Transcribed but not translated Poly-A ➔ Contain AU-rich elements ➔ Added after transcription by poly-A polymerase AU-Rich Elements (ARE) (Figure 15) ➔ Facilitate mRNA degradation through the ➔ Regulates stability of mRNA shortening of poly-A tail ➔ Degradation of poly-A tail is done by exosomes ALTERNATIVE SPLICING _______________________________________________________ Splicing ➔ Involves removal of introns and joining of exons in pre-mRNA ➔ In this stage, pre-mRNA is still in the nucleus After splicing, mRNA will leave the nucleus and genes will be translated in ribosomes Alternative Splicing ➔ A regulatory mechanism for gene expression at RNA processing level ➔ Splicing together different combinations of exons can generate different kind of mRNAs and proteins from the same gene originally Figure 17: Degradation of poly-A tail BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 6 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Some sequences in UTR increase length of poly-A tail → stability → chance of translation ○ Hu proteins control stability of proteins of the mRNA that encodes for the following activities Stops division of neuronal precursor cells Initiate neuronal differentiation SELECTIVE LOCALIZATION _______________________________________________________ mRNAs have specific location in the embryo ○ Ex: Diffusion and local anchoring of nanos mRNA mRNAs assigned for abdomen formation are Figure 20: Active transport along cytoskeleton of mRNA transported to posterior end of the oocyte They bind to proteins in the posterior end RNA INTERFERENCE _______________________________________________________ Proteins expressed from mRNAs stay in the Interfering RNAs inhibit gene expression through gene posterior end silencing ○ Either degrade mRNA or inhibit translation ○ Two important types of regulatory RNA: microRNA (miRNA) ➔ A double-stranded short RNA sequence (21-24 nucleotides long) with no cap or tail Short interfering RNA (siRNA) ➔ A double-stranded short RNA sequence (also 21-24 nucleotides long) similar to miRNA Figure 18: Diffusion and local anchoring of nanos mRNA ○ Ex: Localized protection of heat shock protein mRNAs They stay at the posterior end since proteins in that area protect them from degradation Enzymes that degrade the mRNA are found in other parts of the cells Results in proteins translated from the mRNA being found in the posterior region only Figure 19: Localized protection of hsp83 mRNA ○ Ex: Active transport along cytoskeleton of mRNAs Figure 21: Mechanism of miRNA and siRNA The mRNAs are transported to one part of the embryo by microtubules Specifically, mRNAs are carried by motor proteins that walk along the microtubules Motor proteins direct the mRNA to the region where it should be expressed BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 7 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC DIFFERENTIAL GENE EXPRESSION BIO 130 LEC INTARMED 2030 | Prof. Bordallo/Leonardo | LU2 SEM 1 | SY. 2024-2025 Production of miRNA and siRNA POST TRANSLATIONAL REGULATION ○ miRNA precursors Come from RNAs transcribed in the nucleus RNAs have complementary sequences = form Post Translational Regulation a hairpin loop ➔ Controls gene expression by deciding on the fate Ends of RNA with the loop is cleaved by of protein sequence (e.g. activity of the protein) nuclease, forming the miRNA precursor Precursor is transported to the cytoplasm Some proteins have to be modified through the ○ siRNA precursors following processes before they can function: Come from viruses that have infected the cell ○ Cleavage or RNA produced in the laboratory ○ Transport to final destination Double stranded RNA forms the siRNA Ex: Some proteins are synthesized in the precursor cytosol but only function after transport to ○ miRNA and siRNA associates with DICER mitochondria ◆ A complex of endoribonuclease proteins that ○ Assembly of subunits cleave miRNA and siRNA precursors Ex: The 4 subunits of hemoglobin must be ◆ Forms the functional miRNAs and siRNAs settled before protein performs functions (21-24 nucleotides) ○ Binding of ions ○ Covalent modification miRNA and siRNA associates with RISC Ex: Some proteins are active when RNA-induced silencing complex (RISC) phosphorylated, acetylated, or glycolized ➔ Degrades one of the strands of miRNA or siRNA (through an argonaute protein) ➔ Looks for mRNA complementary to the remaining strand ○ siRNAs: more precise targeting; miRNAs: only a small part binds to mRNA Imprecise matching in miRNAs allow it to target hundreds of endogenous mRNAs ○ If bases are complementary: mRNA is degraded ○ If bases are less of a match: translation is blocked Figure 22: Regulation of maternal mRNAs through miRNA miRNA in the regulation of maternal mRNAs ○ Maternal mRNAs function in controlling development through cleavage ○ However, as the gastrula forms, zygotic genes must be transcribed ○ Hence, miRNA degrades maternal mRNAs during the transition to the gastrula stage BIO 130 LEC LU 2 SEM 1 | IMED 2030 Page 8 of 8 SAMSON, SDAV; AGSALOG, JNM; FABREGAS, MLH; REYES, JCDC