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Lecture 9

Lecture 9

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Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 September 26 2012 Lecture 9 Dr. Hekimi Insulators, also known as “Boundary Elements”, are DNA sequences that prevent eukaryotic gene regulatory proteins from influencing distant genes:  They can block enhancer and silencer elements in a position dependant manner  In pairs, they can create independent functional domains by blocking positive or negative effects of surrounding chromatin  They bind specialized proteins that mediate these effects  Barrier elements block the linear spread of silenced chromatin  Enhancer blocking elements interfere with enhanced transcription o In the example here, the insulator elements prevents the enhancer sequence from affecting gene A, but does not prevent it from activating gene B Insulators can come in three types:  Barrier insulators prevent spreading of heterochromatin and therefore allow gene transcription to occur at a specific region  Enhancer blocking insulators prevent and enhancer sequence from activating a gene across the insulator sequence, thus blocking transcription  Insulated domain insulators essentially isolate a specific region of DNA and prevent it from being affected by both heterochromatin spreading and enhancers that are outside of the insulated domain Barrier insulators can be thought of in terms of heterochromatin and euchromatin:  Heterochromatin: o Closed o Ordered nucleosome arrays o Hypoacetylated core histones o Lack of methylation of H3 K4 and enriched methylation of H3 K9 o Associated with specific chromodomain containing proteins leading to chromatin condensation (and transcription inhibition) o At the center of both initiation and maintenance of heterochromatic structures is a self- perpetuating cycle:  Methylation of H3K9 leads to HP1 mediated recruitment of additional histone methyltransferases (HMTs)  Euchromatin: o Open o Lack of evenly spaced nucleosomes; depletion of histone 1 across the entire domain o Acetylated core histones o Methylation of H3K4 1 th BIOL 300 September 26 2012 Lecture 9 Dr. Hekimi HP1 mediates heterochromatin formation:  HP1 contains a chromodomain that binds to methylated K9 and a chromoshadow domain (named because it is found largely in proteins with chromodomains) that binds to domains of other HP1 molecules (i.e. multimerize) o HP1 proteins can bind to each other to form a higher order structure this allowing the formation of condensed heterochromatin  There is NO HP1 in yeast Heterochromatin condensation can spread along a chromosome because HP1 binds an HTM that methylates H3K9  This is like a feed forward mechanism in which the HMT allows binding of another HP1 on the adjacent nucleosome, which it turn calls in another HMT  The barrier insulator is an insulator DNA sequence that is able to stop the spread of heterochromatin through recruitment of specific proteins with established barrier functions In the absence of a barrier insulator, the extent of heterochromatin propagation is determined by a local balance of activities:  A heterochromatic region will have high amounts of HDACs, H3K9 HMTs as well as HP1, all favoring chromatin condensation  A euchromatic region will be high in HATs, H3K4 HMTs and low in HP1  In this case, the heterochromatin is able to propagate because of the feed-forward mechanism of HP1 o Recall the H3 N terminal tail and all the mechanisms that can work either to favor or inhibit propagation of heterochromatin  E.g. Methylation of K9 would favor binding of HP1 and therefore favor propagation  K4 methylation blocks the actions of proteins like NuRF and SUV39 which prevents propagation Position effect variegation, or PEV, involves insertion of genes into different regions of the genome, either enhancing or suppressing a certain phenotype (for example, Drosophila eye color) 2 th BIOL 300 September 26 2012 Lecture 9 Dr. Hekimi  E.g. the translocation of a gene for eye pigment color into a barrier between heterochromatic and euchromatic regions will cause variegated eye color (some cells will express is, some won’t) Transgenesis is experimental insertion of genes into an organism’s genome; is this gene is inserted without insulators:  If a new gene is inserted into a heterochromatic region by, for example, a transposon, then there will be little to no expression of the gene  However, if the gene is inserted into a euchromatic region of the genome, then transcription will happen at a high level  Insulators are able to overcome the position effect: o Inserting the gene flanked by insulators will protect the surrounded DNA from being condensed and therefore the gene will always be expressed  This essentially creates an insulated DNA domain when it is inserted into a genome Mechanism of barrier action:  Tethering to fixed nuclear structures: o Artificial tethering to a fixed structure such as the NPC can form a loop which is an efficient barrier to the spread of histone modifications and heterochromatin factors associated with repression  Nucleosome gap: o Multiple binding sites for some barrier proteins have been shown to exclude nucleosomes (thus breaking the feed forward mechanism of proteins like HP1 and H3K9 HMT)  Nucleosome masking o Barrier proteins can compete with heterochromatin propagation by binding directly to histones (i.e. directly blocking access to proteins like HP1)  Histone code manipulation: o Histone modifications associated with activation in the immediate vicinity of the barrier can compete with the propagation of heterochromatin factor binding o I.e. If you have more H3K9 acetylation than methylation at a given region, propagation of heterochromatin will be blocked (prevents binding of HP1) Insulators and enhancer-promoter selectivity:  Enhancers
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