Gene regulation at DNA level

I. Introduction 3 II. Regulation of chromatin structure 3 1. Histone modifications 4 1.1 Histone acetylation 4 1.2 Histone phosphorylation 5 1.3 Histone methylation 5 a. Lysine methylation 5 b. Arginine methylation 5 2. DNA methylation 6 2.1 Epigenetic Inheritance 6 III. Noncoding RNAs controlls gene expression by remodeling chromatin 7 1. Small noncoding RNAs 7 2. piRNA 7 3. Long noncoding RNAs 8 IV. Conclusion 10 REFERENCES 11

ĐẠI HỌC QUỐC GIA THÀNH PHỐ HỒ CHÍ MINH ĐẠI HỌC KHOA HỌC TỰ NHIÊN  BÁO CÁO SEMINAR MÔN SINH HỌC PHÂN TỬ ĐẠI CƯƠNG Tên đề tài: GENE REGULATION AT DNA LEVEL Nhóm thực hành: Nhóm Lớp: Sinh học phân tử đại cương CLC2 INDEX I Introduction .3 II Regulation of chromatin structure Histone modifications 1.1 Histone acetylation 1.2 Histone phosphorylation 1.3 Histone methylation a Lysine methylation b Arginine methylation DNA methylation 2.1 Epigenetic Inheritance III Noncoding RNAs controlls gene expression by remodeling chromatin .7 Small noncoding RNAs piRNA Long noncoding RNAs IV Conclusion 10 REFERENCES .11 DISCUSSION .12 I Introduction Our bodies include a variety of cell types that serve a variety of purposes, but despite this, they all have the same genome How did this take place? Gene expression provides the answer to this question We'll start by understanding what gene expression means Most genes have enough information required for transcription and translation to result in the production of proteins with various functions In other words, gene expression is the process by which information travels through the encoded gene to produce RNA and protein with various roles The process of gene expression is intricate and subject to both internal and external influences Compared to prokaryotes, eukaryotes have a far more complex mechanism But there are three key stages that prokaryotes and eukaryotes both go through The gene that facilitates RNA synthesis is the first The polypeptide chain is created once the RNA has been translated into amino acid sequences Finally, these polypeptide chains will curl up and transform into proteins with specific roles The right moment and the appropriate function are the most important things of gene expression regulation So in our presentation, we will discuss how the eukaryotic cells control the expression of genes at DNA level And what is the function of noncoding RNAs in regulating gene expression and how does it relate to the regulation of chromatin structure? II Regulation of chromatin structure Chromatin is a complex organization consisting of DNA strands around histone proteins Histones pack and organize DNA into 30nm structural units known as nucleosome complexes These structures can limit the access of proteins to DNA regions, which in turn controls the structure of chromatin Chromatin is not only an inert structure for packaging DNA into a compact form that stores inside the nucleus, but also is an instructive DNA scaffold that can respond to external cues to regulate gene expression in several ways Both the histone proteins of the nucleosomes that wrap DNA around them and the nucleotides that make up that DNA can undergo chemical modifications (phosphate, methyl, acetyl groups) that affect chromatin structure, but they not alter the DNA base sequence, thus regulating gene expression Histone modifications An insight into how histone modifications could affect chromatin structure came from solving the high-resolution X-ray structure of the nucleosome in 1997 According to the structure, highly basic histone amino (N)-terminal tails have the ability to protrude from their own nucleosome and make contact with nearby nucleosomes Chemical modification of these tails would affect inter-nucleosomal interactions and thus affect the overall chromatin structure, then play a direct role in the regulation of gene transcription These histone tails not regulate chromatin structure by merely being there, but rather than they recruit remodeling enzymes that catalyze the addition or removal of specific chemical groups, such as acetyl (—COCH3), methyl, and phosphate groups 1.1 Histone acetylation It has been demonstrated that the acetylation of lysines is very dynamic and controlled by the antagonistic actions of two groups of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDAC) The transfer of an acetyl group to the ε-amino group of lysine side chains is catalyzed by the HATs, which use acetyl CoA as a cofactor They achieve this by neutralizing the positive charge from the lysine, which may make it more difficult for histones and DNA to connect HDAC enzymes counteract the actions of HATs and undo lysine acetylation, which brings back the amino acid's positive charge As a result, the local chromatin architecture may be stabilized, which is consistent with HDACs' major role as transcriptional repressors 1.2 Histone phosphorylation The phosphorylation of histones is a very dynamic process, just like histone acetylation Serines, threonines, and tyrosines are involved, primarily but not solely in the N-terminal histone tails, where it occurs Kinases and phosphatases, which add and remove the modification, respectively, regulate the modification's levels All of the known histone kinases transfer a phosphate group from ATP to the hydroxyl group of the target amino-acid side chain By doing so, the alteration significantly increases the histone's negative charge, which unquestionably affects the chromatin structure The functions of histone phosphatases are less clear 1.3 Histone methylation Lysine’s and arginines' side chains are the major sites of histone methylation Histone methylation does not affect the charge of the histone protein, in contrast to acetylation and phosphorylation a Lysine methylation The first histone lysine methyltransferase (HKMT) to be found was SUV39H1 which targets H3K9 20 Since then, many HKMTs have been discovered, and the bulk of them methylated lysines in the N-terminal tails Remarkably, a region known as the SET domain, which houses the enzymatic activity, is present in all HKMTs that methylate Nterminal lysines In any instance, the transfer of a methyl group from Sadenosylmethionine (SAM) to a lysine's -amino group is catalyzed by all HKMTs b Arginine methylation The type-I and type-II enzymes are the two kinds of arginine methyltransferase Together, the two varieties of arginine methyltransferases make up the PRMTs, a relatively large protein family with 11 members These enzymes all work with a range of substrates to transfer a methyl group from SAM (S-adenosylmethionine) to the ωguanidino group of arginine DNA methylation It is also possible to alter the DNA molecule itself This takes place in very particular areas known as CpG islands In the promoter regions of genes, there exist sequences with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) When this structure is present, the pair's cytosine member may be methylated (a methyl group is added at C-5) This modification affects how proteins, notably the histone proteins that regulate access to the area, interact with the DNA Deacetylated histones and highly methylated (hypermethylated) DNA sections are tightly coiled and transcriptionally inactive The tissue-specific gene silencing and allele-specific inactivation of the X-chromosome, which are linked to hypermethylation of CpG islands, both have repressive DNA methylation as a characteristic 2.1 Epigenetic Inheritance While the DNA sequence is unaffected by those modifications we just discussed, the pattern of gene expression is handed down to the following generation These changes to DNA are transferred from parent to offspring The transmission of traits through mechanisms other than the nucleotide sequence itself is referred to as epigenetic inheritance The nucleotide sequence is unchanged and the modifications to the DNA and histone proteins are temporary Instead, these modifications affect the chromosomal architecture (open or closed) and are transient (although they frequently last through numerous rounds of cell division) For instance, during gamete creation, DNA methylation patterns are mainly eliminated and then restored during embryonic development III Noncoding RNAs controlls gene expression by remodeling chromatin In recent times, biologists have researched and calculated to point out that just 1,5% of the human genome are protein-coding genes, just a very small quantity of human’s genome Some biologists and scientists have called non-coding protein DNAs "junk DNA", because those DNAs have special roles in synthesizing other RNAs besides mRNA, which could be tRNA, and rRNA Those RNA are called non-protein coding RNA or noncoding RNAs, ncRNAs Noncoding RNAs are classified based on their length Small noncoding RNAs are less than 200 nucleotides in size and include both small interfering RNAs (siRNAs) and microRNAs (miRNAs) Transcripts larger than 200 nucleotides are called long noncoding RNAs (lncRNAs) Small noncoding RNAs In addition to regulating mRNAs, small noncoding RNAs can cause the remodeling of chromatin structure In the S phase of the cell cycle, for example, the centromeric regions of DNA must be loosened for chromosomal replication and then recondensed into heterochromatin in preparation for mitosis In some yeasts, siRNAs produced by the yeast cells from the centromeric DNA are required to re-form the heterochromatin at the centromeres Exactly how the process starts is still debated, but biologists agree on the general idea: The siRNA system in yeast interacts with other, larger noncoding RNAs and chromatin-modifying enzymes to condense the centromere chromatin into heterochromatin In most mammalian cells, siRNAs have not been found, and the mechanism for centromere DNA condensation is not yet understood However, it may also involve small noncoding RNAs piRNA PiRNAs are small ncRNAs of 24–31 nucleotides in size named for their ability to form complexes with Piwi proteins of the Argonaute family piRNAs have a function in inducing the formation of heterochromatin, blocking the expression of some parasitic DNA elements in the genome known as transposons Transposons are nucleic acid sequences in DNA that can change their position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size The primary role of these small RNAs is suppression of transposon activity during germ line development More than 90% of mammalian piRNAs map uniquely in the genome and cluster to a small number of loci However, transposon control also occurs in mammals during spermatogenesis through de novo DNA methylation Long noncoding RNAs Researchers have also found a relatively large number of long noncoding RNAs (lncRNAs), ranging from 200 to hundreds of thousands of nucleotides in length, that are expressed at significant levels in specific cell types at particular times LncRNAs have been proposed to regulate transcription by recruiting chromatin-remodeling complexes, which in turn mediate epigenetic changes The repressive PcG is one of the most welldescribed transcriptional complexes that initiate and maintain epigenetic changes PcG is characterized as two multiprotein complexes—polycomb repressive complex (PRC1) and (PRC2) Components of PRC2 trimethylate H3K27, establishing the silent chromatin state Components of PRC1 bind H3K27me3 and ubiquitinate lysine 119 on histone 2A Interestingly, components of PRC1 and PRC2 are also RNA-binding proteins One lncRNA, long known to be functional, is responsible for X chromosome inactivation, which prevents the expression of genes located on one of the X chromosomes in most female mammals In this case, lncRNAs—transcripts of the XIST gene located on the chromosome to be inactivated—bind back to and coat that chromosome This binding leads to the condensation of the entire chromosome into heterochromatin The examples just described involve chromatin remodeling in large regions of the chromosome Because chromatin structure affects transcription and thus gene expression, RNA-based regulation of chromatin structure is sure to play an essential role in gene regulation Additionally, an alternate role for lncRNAs in which they can act as a scaffold, bringing DNA, proteins, and other RNAs together into complexes These associations may act either to condense chromatin or, in some cases, to help bring the enhancer of a gene together with mediator proteins and the gene’s promoter, activating gene expression more directly Mechanisms for regulation of epigenetics and gene expression by non-coding RNAs NcRNAs can function as chromatin remodeling in regulating gene expression (D through F) (D) LncRNA transcribed from the minor promoter of dihydrofolate reductase (DHFR) forms a triplex together with the transcription factor TFIIB and the major promoter leading to the dissociation of the preinitiation complex (E) Enhancer region (i and ii) of Dlx5/6 generates a lncRNA Evf-2 which forms a complex with homeodomain protein Dlx-2 to activate transcription (F) Transcription of B2 and Alu RNAs is induced upon heat shock They inhibit mRNA synthesis by disrupting contacts between RNA polymerase II and promoter DNA IV Conclusion In conclusion, regulation of gene expression at DNA level is undoubtedly important to living creatures We wondered how different cell types share the same genome and what gene expression is We answered the question through the regulation of chromatin structure which relates to the regulation of gene expression In detail, the recruitment of proteins and complexes with specific enzymatic activities modify chromatin structure What’s more, we found out that gene expression is controlled by noncoding RNAs such as: small noncoding RNAs, piRNA and long noncoding RNAs These noncoding RNAs remodel chromatin structure, blocking the expression of transposons and regulating transcription Their function is mainly to keep the process of gene expression going smoothly without any flaws That's why gene expression has been considered a highly accurate process Regulation of gene expression at DNA level can be affected by several factors We can control it to our desired result as long as we understand how it works 10 REFERENCES [1] Regulation of chromatin structure - control of gene expression in eukaryotes - MCAT content (2021) Jack Westin Available at: https://jackwestin.com/resources/mcat-content/ control-of-gene-expression-in-eukaryotes/regulation-of-chromatin-structure (Accessed: February 14, 2023) [2] Bannister, A.J and Kouzarides, T (2011) “Regulation of chromatin by histone modifications” Cell Research, 21(3), pp 381–395 Available at: https://doi.org/10.1038/cr.2011.22 [3] Kaikkonen, M U., Lam, T Y., & Glass, C K (2011) Editor's Choice: Non-coding RNAs as regulators of gene expression and epigenetics Cardiovascular Research, 90(3), 430 https://doi.org/10.1093/cvr/cvr097 [4] Lisa A Urry, Michael L Cain, Steven A Wasserman, Peter V Minorsky, Rebecca B Orr, Neil A Campbell Campbell biology Twelfth edition | New York, NY: Pearson, 2020 At Unit 3, chapter 18, concept https://lccn.loc.gov/2019039139 11 18.3, page 379 Available at DISCUSSION Question 1: What controls the histone modification in recognition of which chromatin regions should be heterochromatin or euchromatin? Answer: Histone acetyltransferases and histone deacetylases recognize specific DNA sequences such as CpG islands, GC-rich regions, and AT-rich regions to indicate whether a chromatin region should be euchromatin or heterochromatin GC-rich regions are often associated with euchromatin regions, while GpG islands and AT-rich regions are more likely to be associated with heterochromatin These specific DNA sequences act as markers for the histone acetyltransferases and deacetylases that recognize them and indicate whether a chromatin region should be euchromatin or heterochromatin According to a scientific paper[1], It appears that methyl-CpG binding proteins may have an impact on histone modification in mammals This influence is likely due to their connections with modification complexes that are capable of either deacetylating or methylating nucleosomes near methylated DNA Another research [2] making a comparison about GC- and AT-rich isochores of yeast have shown that GC- and AT-rich isochores differ in chromatin structure, with more open and more compact chromatin in the two types of regions, respectively Additionally, studies of yeast isochores by 3C (chromosome conformation capture) analysis have revealed important structural differences Chromatin in AT-rich isochores has a longer apparent persistence length than that in GC-rich isochores, suggesting that AT-rich chromatin is less flexible than GC-rich chromatin References: 12 [1]: Gartler, S.M., Varadarajan, K.R., Luo, P et al Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins BMC Biol 2, 21 (2004) https://doi.org/10.1186/1741-7007-2-21 [2]: Dekker, J GC- and AT-rich chromatin domains differ in conformation and histone modification status and are differentially modulated by Rpd3p Genome Biol 8, R116 (2007) https://doi.org/10.1186/gb-2007-8-6-r116 Question 2: The meaning of histone modification in DNA replication? Answer: DNA replication also affects the structure of chromatin The replication machinery first enlists particular histone modifiers to change histones at replication origins In addition, when replication proceeds, old histones are shed and new ones are created to wrap DNA Histones that have just been created have specific H3 and H4 residues that are acetylated, but they lack positional information These histones can undergo immediate postreplication alteration or delayed modification In order to integrate the action of several DNA-related activities, most notably gene expression and DNA replication, the patterns of histone modifications H3K27me3 is mainly found in repressive areas, H3K56ac is deposited on newly duplicated DNA, and H3K4me3 and H3K9ac are associated with gene expression Certain histone modifications may be linked to processes like DNA damage repair or replication in addition to transcription and replication alone As all these impacts are frequently combined in histone modification profiles, it might be challenging to determine how each activity contributed Signals present on both the DNA and the histone proteins influence how the histone proteins move These signals are tags that are attached to DNA and histone proteins to inform the histones whether a chromosomal region should be open or closed These tags are not fixed; they can be changed or eliminated as necessary These are chemical alterations (phosphate, methyl, or acetyl groups) connected to particular amino acids in 13 the protein or to the DNA nucleotides Although the tags don't change the DNA's base sequence, they change how tightly the DNA is twisted around the histone proteins Because the DNA molecule is negatively charged, variations in the charge of the histone will alter how tightly the DNA molecule is wound The histone proteins have a strong positive charge when they are unaltered; this charge is reduced by the addition of chemical modifications, such as acetyl groups Modifications can also be made to the DNA molecule itself CpG islands are highly particular geographic areas where this occurs These are sections of the promoter regions of genes where cytosine and guanine dinucleotide DNA pairs (CG) occur frequently The cytosine in the pair can be methylated when this arrangement is present (a methyl group is added) As histone proteins regulate access to the area, this alteration alters how the DNA interacts with these proteins References: Bar-Ziv R, Voichek Y, Barkai N Chromatin dynamics during DNA replication Genome Res 2016 Sep;26(9):1245-56 doi: 10.1101/gr.201244.115 Epub 2016 May 25 PMID: 27225843; PMCID: PMC5052047 Question 3: Is there any structure that can replace nucleosome in regulating transcription? Answer: - The Histone Amino Terminal Tails There could be hundreds of different nucleosomal charge distribution states in vivo as a result of various combinations of acetylation patterns on the eight tails in each nucleosome This is because acetylation of multiple sites on each of the eight histone monomers in a nucleosome would significantly alter the overall charge of the nucleosome Hence, it doesn't seem that these tails interact in a static way with the particle in which they are present Instead, they might interact with other proteins or 14 DNA sequences in a way that alters the nucleosome's ability to function Characterizing nucleosomes with higher degrees of acetylation or nucleosomes that have undergone trypsin treatment to remove the tails has allowed researchers to better understand the function of the N termini - ATP- dependent remodeling complexes They seem to increase access to the nucleosome's DNA without eliminating histones A molecular route that results in a stable change in chromatin structure may start with the capacity of chromatin remodeling complexes to catalyze the creation of an altered nucleosomal state One of the changed nucleosome states that have been observed by varied solution conditions may mimic the conformation of nucleosomes that have been modified by these ATP-dependent remodeling activities These biophysical findings highlight the fact that nucleosome structure can change over time in solutions, giving credence to the idea that these complexes might employ the energy released during ATP hydrolysis to force the nucleosome into a different configuration References: [1]: J L Workman, R E Kingston Alteration of nucleosome structure as a mechanism of - annual reviews (n.d.) Retrieved March 6, 2023, from https://www.annualreviews.org/doi/10.1146/annurev.biochem.67.1.545 Question 4: How does small non-coding RNA affect euchromatin and heterochromatin through gene regulation? Answer: sncRNAs can affect euchromatin and heterochromatin is through their interaction with the RNA-induced transcriptional silencing (RITS) complex The RITS complex contains sncRNAs, including small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), and can bind to specific regions of chromatin, leading to changes in gene expression 15 For example, model for heterochromatin assembly and spreading at S pombe centromeric outer repeats Heterochromatic centromere sequences (yellow arrow) are transcribed by RNA Polymerase II These centromere transcripts are targeted by RITS via siRNA-loaded Ago1 The association of RITS with centromere heterochromatin is strengthened by the binding of Chp1 to H3mK9 RITS activity can recruit both CLRC, via interactions with Stc1, and RDRC resulting in the spreading of H3mK9 and amplification of siRNAs, respectively dsRNA generated either by bi-directional transcription from centromere promoters (black arrows) or by RDRC activity is recognized and processed by Dicer (Dcr1) The resulting centromere siRNAs are then loaded onto Ago1 first in the ARC complex and then in RITS snc-RNA involves in gene expression Overall, sncRNAs play important roles in gene regulation by interacting with chromatin and modulating the expression of genes in chromatin modification References: Volpe T, Martienssen RA RNA interference and heterochromatin assembly Cold Spring Harb Perspect Biol 2011 Sep 1;3(9):a003731 doi: 10.1101/cshperspect.a003731 PMID: 21441597; PMCID: PMC3181039 16