Chromatin and Transcription and Imprinting

Classic experiment on role of nucleosomes in transcription

-          take piece of DNA with txn start site and GAL4 binding site

o       in one tube, assemble nucleosomes on the DNA, then measure txn with/without GAL4p when basal factors/RNAPII/rNTPs are present

o       in other tube, leave DNA naked, measure txn with/without GAL4p when basal factors/RNAPII/rNTPs are present

§         find that tube with naked DNA has much more txn in absence of GAL4p than tube with DNA in nucleosomes

§         when add GAL4p in either tube, get about same amt of txn

o       conclusion – nucleosomes repress basal txn (makes fold activation of activator proteins much larger)

o       if add H1 to the template with nucleosomes, repression is even more

Chromatin remodeling

-          can modify histones by covalent modifications

o       acetylation of histones on lysines makes the histone less positively charged, binds DNA less well, allows RNAP etc to bind DNA

o       deacetylation of histones reverses all this, makes the chromatin more condensed, shuts down txn

-          there are protein complexes that “remove” histones from DNA

o       SWI/SNF and SAGA

§         These are machines that use energy (ATP) to remove or remodel nucleosomes along DNA

§         Makes it easier to do txn

§         These are recruited to DNA by transcriptional activator proteins

-          Nucleosome-free regions

o       Some txn control regions (promoter proximal elements and enhancers) remain nucleosome-free all the time

§         Can see as a DNaseI hypersensitive site

·         This enzyme has access to DNA when nucleosome not there, it is blocked by nucleosomes

§         Presumably makes it easier to regulatory proteins to bind to their sites in the PPEs or enhancers

Hypercondensation

-          when DNA is methylated, it is usually associated with very highly condensed chromatin

o       methylation happens at CG dinucleotides

o       because there is an enzyme that recognizes that sequence and methylates the C

o       causes chromatin to become highly condensed

§         a protein called MECP2 binds to methylated regions and appears to be involved in this

·         mutations in MECP2 cause Rett syndrome, an autism-like disorder

o       in about 1% of autism patients there is a MECP2 mutation

·         what would be effect on txn if you lacked MECP2?

·         Exciting experiment – mice that lack MECP2 show signs of this disease – if express MECP2 later in life it seems to reverse the disease

Imprinting

-          if take enucleated egg and add a sperm nucleus and an egg nucleus, develops into viable, fertile, adult mouse

-          if put in two sperm nuclei or two egg nuclei, they don’t develop normally

o       suggests that the two sets of chromosomes not really identical

-          Igf-2 gene and imprinting

o       If dad sperm has deletion of this gene and mom egg has gene, mice develop small

o       If mom egg has deletion of this gene and dad sperm has gene, mice develop normally

§         Model is that maternal version of gene is silenced, but dads is actively transcribed

-          There are some other genes in mammals (only), about 500? or so, that show this phenomenon – genomic imprinting

o       The gene has no mutation, but it is simply not expressed

o       Epigenetic – outside the genes, but heritable

§         Seems to be a relationship to methylation – the methylated version of the gene is silenced

-          H19 gene

o       Imprinted the opposite way of Igf-2 gene

§         Not transcribed in males, is transcribed in females

o       Turns out it is close on chromosome to Igf-2 gene

o       Fig. 18.15d shows model

§         Insulator between these genes – when inactivated by methylation the enhancer stimulates txn only from Igf-2 gene, but when insulator is active (binding CTCF protein) only H19 is active

-          Imprinting and human genetic disease

o       Prader-Willi and Angelman syndromes

§         Both from deletion of bit of chr. 15

§         Depends whether the deletion comes from sperm or egg

§         If deletion inherited from father, child has Prader-Willi

§         If deletion inherited from mother, child has Angelman

·         Must be (at least) two imprinted genes

o       One maternally imprinted (inactive) – if receive deletion from dad and imprinted (inactive) from mom, get Prader-Willi

o       One paternally imprinted (inactive) – if receive deletion from mom and imprinted (inactive) from dad, get Angelman

-          Imprinting and evolution

o       Only mammals have imprinting and only mammals develop in a placenta

§         Could be competition in utero between embryos, for species that can mate with multiple males and have all developing at same time

Problems from chapter 18 so far……

2b, 3, 4, 5, 6, 7, 8, 9, 13, 14, 16a, 18a b, 19, 20, 21, 23