regulation of transcription in eukaryotes

1. • HIGHER EUKARYOTES HAVE
MORE GENES.
• EXPRESSION IS DIFFERENT IN
DIFFRENT TISSUES AND AT
DIFFRENT STAGES OF
DEVELOPMENT.
• FOR TRANSCRIPTION DNA
SHOULD BE EXPOSED.
• EXPRESSION REQUIRES
ACTIVATORS AND
ENHANCERS.
• T-FACTORS SHOULD BE
TRANSPORTED TO THE
NUCLEUS.

2. PROPERTIES.
• RESPOND TO A SPECIFIC STIMULUS.
• CAPABLE OF ENTERING THE NUCLEUS.
• RECOGNISE & BIND TO SPECIFIC DNA SEQUENCE.
• DIRECT/INDIRECT CONTACT WITH T-APPARATUS.
• T-FACTORS HAVE TWO DOMAINS : 1ST THAT BINDS TO DNA & 2ND
THAT INTERACTS WITH T-APPARATUS

3.  Mediator: protein complex that sits on the top of RNA Pol II & provides site
of contact for activators (grant RNA pol permission to proceed forward from
promoter ; contact with general T-factors – TFIIB TFIID TFIIH)
 Mediator contains 20 protein subunits & constant core (attached to this are
other subunits that vary between organisms & also between different tisues
within same organism)
 It combines & receives signals from multiple activators/repressors & send
result to RNA Pol II enzyme
 Some subunits of mediator act in a positive manner while others act in a
negative manner.
 Individual mediator proteins :- ‘co-activator proteins’

4.  Enhancers loop around DNA so that activator proteins bound at enhancer
can make contact with T-apparatus via mediator complex
How enhancer is prevented from activating genes further along
the chromosome ??
 Chromosomes are divided into regulatory neighborhoods by special
sequences : ‘boundary elements’ (bcz they form boundaries to the regions
of heterochromatin) or ‘INSULATORS’
 Insulators are regions of DNA that bind to ‘ZINC FINGERS’ (also called
insulator binding proteins ; IBP’s) & these are rich in GC regions & CG
sequences may be methylated.
 Eg: in vertebrates, CTCF (CCCTC-binding factors) must bind to insulator to
block enhancers
 When insulator is methylated, it dsnt bind to CTCF & no longer functions
 Insulators prevent enhancers from interfering with the wrong genes & also
prevent the spread of heterochromatin.
 Insulators : fixed/mobile (gypsy element ;a retrotransposon)

5.  During interphase,a filamentous web of proteins (nuclear matix)
appears inside nuclear membrane ; DNA is attached to these
proteins by sites called MARs (also called SARs i.e. scaffold
attachment regions) . These protein bound MARs sites recognise
the bent DNA (DNA with multiple runs of A’s is bent)
 MARs : 200-1000 bps long, AT-rich
 Next to MARs are ‘topoisomerase II recognition sites’
 Chromatin remodeling also starts from MARs site
 Eg : in transgenic animals, the efficient expression of transgene is
helped my making sure that it lies between two MAR sites bcz this
region is more likely to be opened up for transcription.

6. Looped domains between MAR sites
Alhough many loops are present but only a single loop of histone
free DNA is drawn coming from a region of the nuclear scaffold.
MARs contain MAR proteins that anchor DNA to the scaffold.

7.  Negative regulators act by occupying the recognition site of activator on
DNA ti prevent its binding.
 Eg: activation of H2B gene requires binding of activator CTF to CAAT
sequence. But CDP (CAAT displacement protein) can also occupy CAAT
box & prevent binding activator thereby preventing the assembly of T-
apparatus but CDP dsnt block the binding site for RNA pol
 Eg: MyoD (T-factor needed for the formation of muscle cells & member of
bHLH proteins : DNA binding proteins that bind DNA as dimers; bHLH
function as heterodimers(tissue specific bHLH + widely spread E protein)
 By itself, MyoD dimerizes poorly. So it forms mixed dimers with E12/E47
(similar in shape & str to MyoD but expressed in all tissues unlike MyoD) .
Id protein binds to MyoD & E12/E47 & inhibits differentiation.

8. Myod: a T-factor
Interference with
activators

9.  Heterochromatin is densely packaged DNA that can’t be transcribed bcz
RNA Pol can’t gain access to promoter
 Histones : H1,(H2A,H2B,H3 & H4: core histones)
 Acetylation of nulceosomes: Core histones can be acetylated (form less
condensed chromatin) & degree of aceytlation affects the state of
nucleosome aggregation & gene expression. Enzymes (HATs, HDACs eg:
human CBP p300 proteins) bind to T-factors
 Chromatin remodeling complexes (CRCs): 1st they slide nucleosomes
along a DNA molecule & exposing seq. for transcription and 2nd ability to
rearrange histones loosening nucleosome structure allowing access to DNA
2 families of CRCs: 1) larger Swi/Snf (switch snuff) complexes
2) smaller ISWI (initiation switch) complexes

11. I. T-factor binds to the DNA
II. HATs binds to the T-factor
III. HAT acetylates the histones & association of
nucleosomes is loosened
IV. CRC slides & rearranges nucleosomes allowing the
binding access to the DNA
V. Binding of T-factors
VI. Binding of RNA pol to the DNA
VII. Initiation requires a positive signal to be transmitted
via the mediator complex from 1 or more specific T-
factors

12. There are 3 types of methylases:
1) Maintenance methylases: add methyl groups to newly made
DNA at locations opp. Methyl groups on old parental DNA strand
(ensuring that pattern of methylation is inherited during
chromosome division)
2) De novo methylases: change the pattern of methylation
3) Demethylases: removes the methyl groups
 Methylation silences gene expression
 About half the genes are loacted close to CG islands. Housekeeping
genes (expressed in all tissues) possess non methylated CG
islands. In contrast, Cg islands of tissue specific genes are non-
methylated only in those particular tissues where the genes are
expressed.

13.  Genes are silenced by methylation of DNA or covalent modifications
of both DNA & histones
 Silencing affects a single gene/cluster of genes/substantial region of
chromosome/whole chromosome
 Methylation of cytosine in CG/CNG sequences occurs where methyl grp
project into the major groove of DNA & hinder the binding of T-factors
 Methylated CG seq. are recognised & bound by MeCPs recognised by
other proteins that remove acetyl groups from histones condensation of
DNA to form heterochromatin
 DNA methylation patterns are repogrammed when a new zygote is
formed : just after fertilisation, methylation pattern of most of the DNA are
erased leading to new patterns in a tissue specific manner
 Those genes whose promoter regions are methylated are silenced.

15.  Imprinting occurs when methylation patterns from the gametes survive the
formation of the zygote & affect gene expression in the new organism.
 A few special genes retain their methylation patterns through fertilisation.
 Imprinting ensures that only one of a pair of genes in a diploid cell is
expressed. The second copy is silenced by methylation. (the choice as to
which allele of a genes to express depends on its parental origin)
 20 imprinted genes are known in mouse. Eg: IGF-II from father is
expressed whereas maternal allele isn’t. Usually, it’s the paternal allele that
is expressed in zygote bt nt always. Eg: of expression of paternal allele is
IGF-IIR (receptor for IGF-II)

16. Special form of imprinting
 C.elegans: expression level on both X-chromosomes(f) is halved. In
males, thr is no Y chromosome ie. Single unpaired X-chromosome
(XO) & XX are hermaphrodites. (sdc2 & msl2)
 Drosophila: expression of genes on the single X-chromosome is
doubled
 Mammals: 1of the pair of X-chromosome in each female cell is
silenced ( X-inactivation by methylation of Xist gene/ non coding
RNA inactivation)
 Worms & insects: protein complexes which dec./inc. transcription of
genes located on X-chromosome