Gene regulation at DNA level

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 3
Lớp: Sinh học phân tử đại cương CLC2

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INDEX
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
DISCUSSION.................................................................................................................12

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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.

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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 do not alter the DNA base
sequence, thus regulating gene expression.
1. 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 do 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.
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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.

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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.
2. 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
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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)
1. 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

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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.
2. 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.
3. 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 1 (PRC1)
and 2 (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
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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
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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.

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REFERENCES
[1] Regulation of chromatin structure - control of gene expression in eukaryotes - MCAT
content (2021) Jack Westin. Available at: />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”

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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:

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[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). />[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). />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
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the protein or to the DNA nucleotides. Although the tags don't change the DNA's base
sequence, they do 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
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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

/>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.

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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.

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