Regulation of gene expression in eukaryotes- Significance and mechanisms

1. Gene regulation is significantly more complex in eukaryotes than in prokaryotes for a number of
reasons-
1) Large Genome
First, the genome being regulated is significantly larger. The E. coli genome consists of a single,
circular chromosome containing 4.6 Mb. This genome encodes approximately 2000 proteins. In
comparison, the genome within a human cell contains 23 pairs of chromosomes ranging in size from
50 to 250 Mb. Approximately 40,000 genes are present within the 3000 Mb of human DNA. It
would be very difficult for a DNA-binding protein to recognize a unique site in this vast array of DNA
sequences. Consequently, more-elaborate mechanisms are required to achieve specificity.
2) Complex Genome
Another source of complexity in eukaryotic gene regulation is the many different cell types present in
most eukaryotes. Liver and pancreatic cells, for example, differ dramatically in the genes that are
highly expressed.
3) Widely spread genes- No well-defined operons
Moreover, eukaryotic genes are not generally organized into operons. Instead, genes that encode
proteins for steps within a given pathway are often spread widely across the genome.
4) Compact Genome
The DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex called
chromatin. Histones are an important part of this complex since they form the structures known as
nucleosomes and also contribute significantly into gene regulatory mechanisms.
5) Uncoupled transcription and Translation
Finally, transcription and translation are uncoupled in eukaryotes, eliminating some potential gene-
regulatory mechanisms.
Mechanism of regulation of gene expression in Eukaryotes
1) Chromatin Remodeling
Chromatin structure provides an important level of control of gene transcription. Large regions of
chromatin are transcriptionally inactive while others are either active or potentially active. With few
exceptions, each cell contains the same complement of genes (antibody-producing cells are a notable

2. exception). The development of specialized organs, tissues, and cells and their function in the intact
organism depend upon the differential expression of genes. Some of this differential expression is
achieved by having different regions of chromatin available for transcription in cells from various
tissues. For example, the DNA containing the β-globin gene cluster is in "active" chromatin in the
reticulocyte but in "inactive" chromatin in muscle cells.
Formation and disruption of nucleosome structure
The presence of nucleosomes and of complexes of histones and DNA certainly provides a barrier
against the ready association of transcription factors with specific DNA regions. The dynamics of the
formation and disruption of nucleosome structure are therefore an important part of eukaryotic gene
regulation and the processes involved are as follows-
i) Histone acetylation and deacetylation is an important determinant of gene activity.
Acetylation is known to occur on lysine residues in the amino terminal tails of histone molecules
(Figure-1). This modification reduces the positive charge of these tails and decreases the binding
affinity of histone for the negatively charged DNA. Accordingly, the acetylation of histones could
result in disruption of nucleosomal structure and allow readier access of transcription factors to
cognate regulatory DNA elements. Different proteins with specific acetylase and deacetylase
activities are associated with various components of the transcription apparatus.
Figure-1- Showing the Acetylation of lysine residues in the amino terminal ends of Histones. The
postive charge is removed after acteylation.
Thus, histone acetylation can activate transcription through a combination of three mechanisms: by
reducing the affinity of the histones for DNA, by recruiting other components of the transcriptional
machinery, and by initiating the active remodeling of the chromatin structure (Figure-2).

3. Figure-2- Acetylation of histones leads to disruption of nucleosomal structure and access
of transcription machinery for transcription of required genes
ii) Modification of DNA-The modification of DNA provides another mechanism, in addition to
packaging with histones, for inhibiting inappropriate gene expression in specific cell types.
Methylation of deoxycytidine residues (Figure-3) in DNA may effect gross changes in chromatin so
as to preclude its active transcription. Acute demethylation of deoxycytidine residues in a specific
region of the tyrosine aminotransferase gene—in response to glucocorticoid hormones—has been
associated with an increased rate of transcription of the gene. However, it is not possible to
generalize that methylated DNA is transcriptionally inactive, that all inactive chromatin is
methylated, or that active DNA is not methylated.
Figure-3- Methylation of deoxycytidine residues in DNA preclude its active transcription.

4. iii) DNA binding proteins- The interactions between DNA-binding proteins such as CAP and
RNA polymerase can activate transcription in prokaryotic cells. Such protein-protein interactions
play a dominant role in eukaryotic gene regulation. In contrast with those of prokaryotic
transcription, few eukaryotic transcription factors have any effect on transcription on their own.
Instead, each factor recruits other proteins to build up large complexes that interact with the
transcriptional machinery to activate or repress transcription.
A major advantage of this mode of regulation is that a given regulatory protein can have different
effects, depending on what other proteins are present in the same cell. This phenomenon,
called combinatorial control, is crucial to multicellular organisms that have many different cell
types.
The binding of specific transcription factors to certain DNA elements may result in disruption of
nucleosomal structure. Many eukaryotic genes have multiple protein-binding DNA elements. The
serial binding of transcription factors to these elements may either directly disrupt the structure of
the nucleosome or prevent its re-formation. These reactions result in chromatin-level structural
changes that in the end increase DNA accessibility to other factors and the transcription machinery.
2) Enhancers and Repressors- Enhancer elements are DNA sequences, although they have no
promoter activity of their own but they greatly increase the activities of many promoters in
eukaryotes. Enhancers function by serving as binding sites for specific regulatory proteins. An
enhancer is effective only in the specific cell types in which appropriate regulatory proteins are
expressed. In many cases, these DNA-binding proteins influence transcription initiation by
perturbing the local chromatin structure to expose a gene or its regulatory sites rather than by direct
interactions with RNA polymerase.
Enhancer elements can exert their positive influence on transcription even when separated by
thousands of base pairs from a promoter; they work when oriented in either direction; and they can
work upstream (5') or downstream (3') from the promoter. Enhancers are promiscuous; they can
stimulate any promoter in the vicinity and may act on more than one promoter.
The elements that decrease or repress the expression of specific genes have also been identified.
Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs
away from the gene they control. However, when transcription factors bind to them, expression of
the gene they control is repressed.
Tissue-specific gene expression is mediated by enhancers or enhancer-like elements. Many genes are
now recognized to harbor enhancer or activator elements in various locations relative to their coding
regions. In addition to being able to enhance gene transcription, some of these enhancer elements
clearly possess the ability to do so in a tissue-specific manner. Thus, the enhancer element associated

5. with the immunoglobulin genes between the J and C regions enhances the expression of those genes
preferentially in lymphoid cells.
3) Locus control regions and Insulators- some regions are controlled by complex DNA
elements called locus control regions (LCRs). An LCR—with associated bound proteins—controls the
expression of a cluster of genes. The best-defined LCR regulates expression of the globin gene family
over a large region of DNA.
Another mechanism is provided by insulators. These DNA elements, also in association with one or
more proteins, prevent an enhancer from acting on a promoter .
4) Gene Amplification- One way to increase the rate at which gene product can be increased is to
increase the number of genes available for transcription of specific molecules. Among the repetitive
DNA sequences are hundreds of copies of ribosomal RNA genes and tRNA genes. These genes
preexist repetitively in the genomic material of the gametes and thus are transmitted in high copy
numbers from generation to generation.
During early development of metazoans, there is an abrupt increase in the need for specific
molecules such as ribosomal RNA and messenger RNA molecules for proteins that make up such
organs as the eggshell. Such requirements are fulfilled by amplification of specific genes.
Subsequently, these amplified genes (Figure-4) presumably generated by a process of repeated
initiations during DNA synthesis, provide multiple sites for gene transcription.
Figure-4- gene amplification increases the copy number of genes and hence increase in the
amount of gene product
In some cases, a several thousand-fold increase in the copy number of specific genes can be achieved
over a period of time involving increasing doses of selective drugs. It has been demonstrated in

6. patients receiving methotrexate for cancer that malignant cells can develop drug resistance by
increasing the number of genes for dihydrofolate reductase, the target of Methotrexate.
5. Gene Rearrangement- Gene rearrangement is observed during immunoglobulins synthesis.
Immunoglobulins are composed of two polypeptides, heavy (about 50 kDa) and light (about 25 kDa)
chains. The mRNAs encoding these two protein subunits are encoded by gene sequences that are
subjected to extensive DNA sequence-coding changes. These DNA coding changes are needed for
generating the required recognition diversity central to appropriate immune function.
IgG heavy and light chain mRNAs are encoded by several different segments that are tandemly
repeated in the germ line. Thus, for example, the IgG light chain is composed of variable (VL), joining
(JL), and constant (CL) domains or segments. For particular subsets of IgG light chains, there are
roughly 250-300 tandemly repeated VL gene coding segments, five tandemly arranged JL coding
sequences, and roughly ten CL gene coding segments. All of these multiple, distinct coding regions
are located in the same region of the same chromosome (Figure-4).By having multiple VL, JL, and
CL segments to choose from, an immune cell has a greater repertoire of sequences to work with to
develop both immunologic flexibility and specificity.
However, a given functional IgG light chain transcription unit contains only the coding sequences for
a single protein. Thus, before a particular IgG light chain can be expressed, single VL, JL, and
CL coding sequences must be recombined to generate a single, contiguous transcription unit
excluding the multiple nonutilized segments (ie, the other approximately 300 unused VL segments,
the other four unused JL segments, and the other nine unused CL segments). This deletion of unused
genetic information is accomplished by selective DNA recombination that removes the unwanted
coding DNA while retaining the required coding sequences: one VL, one JL, and one CL sequence.
(VL sequences are subjected to additional point mutagenesis to generate even more variability—
hence the name.) The newly recombined sequences thus form a single transcription unit that is
competent for RNA polymerase II-mediated transcription.

7. Figure-5- Showing Immunoglobulin m RNA for a light chain formed by transcription of
rearranged genes.
6. Alternative RNA Processing
Eukaryotic cells also employ alternative RNA processing to control gene expression. This can result
when alternative promoters, intron-exon splice sites, or polyadenylation sites are used. Occasionally,
heterogeneity within a cell results, but more commonly the same primary transcript is processed
differently in different tissues.
Alternative polyadenylation sites in the immunoglobulin (Ig M) heavy chain primary transcript
result in mRNAs that are either 2700 bases long (m) or 2400 bases long (s). This results in a different
carboxyl terminal region of the encoded proteins such that the m protein remains attached to the
membrane of the B lymphocyte and the s immunoglobulin is secreted.
Alternative splicing and processing, results in the formation of seven unique -tropomyosin mRNAs
in seven different tissues (Figure-6).

8. Figure-6- The presence or absence of extra exon can alter the structure and hence the functions of a
protein.
7.Class switching- In this process one gene is switched off and a closely related gene takes up the
function.
For example- During intrauterine life embryonic Hb is the first Hb to be formed. It is produced by
having two “Zeta” and two “Epsilon” chains. By the sixth month of intrauterine life, embryonic Hb is
replaced by HbF consisting of “α2 and y2 chains. After birth HbF is replaced by adult type of Hb A
1(97%) and HbA2(3%). Thus the genes for a particular class of Hb are switched off and for another
class are switched on.
Gene switching is also observed in the formation of immunoglobulins. Ig M is the formed during
primary immune response, while Ig G is formed during secondary immune response.
8. mRNA stability- Although most mRNAs in mammalian cells are very stable (half-lives
measured in hours), some turn over very rapidly (half-lives of 10–30 minutes). In certain instances,
mRNA stability is subject to regulation. This has important implications since there is usually a
direct relationship between mRNA amount and the translation of that mRNA into its cognate
protein. Changes in the stability of a specific mRNA can therefore have major effects on biologic
processes.
The stability of the m RNA can be influenced by hormones and certain other effectors.

9. The ends of mRNA molecules are involved in mRNA stability. The 5' cap structure in
eukaryotic mRNA prevents attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3'
exonucleases.
9.Specific motifs of regulatory proteins- Certain DNA binding proteins having specific motifs
bind certain region of DNA to influence the rate of transcription. The specificity involved in the
control of transcription requires that regulatory proteins bind with high affinity to the correct region
of DNA. Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine zipper—
account for many of these specific protein-DNA interactions. The motifs found in these proteins are
unique; their presence in a protein of unknown function suggests that the protein may bind to DNA.
The protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.

10. The ends of mRNA molecules are involved in mRNA stability. The 5' cap structure in
eukaryotic mRNA prevents attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3'
exonucleases.
9.Specific motifs of regulatory proteins- Certain DNA binding proteins having specific motifs
bind certain region of DNA to influence the rate of transcription. The specificity involved in the
control of transcription requires that regulatory proteins bind with high affinity to the correct region
of DNA. Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine zipper—
account for many of these specific protein-DNA interactions. The motifs found in these proteins are
unique; their presence in a protein of unknown function suggests that the protein may bind to DNA.
The protein-DNA interactions are maintained by hydrogen bonds and van der Waals forces.