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Methylation of Histone Proteins at CP

-mechanism that causes repression of transcription
-position and level of methylation determine how histone modifications can lead to repression
-methylation of DNA at CpG islands can also cause repression


Single Competition

-among activators and repressors for binding to DNA
-single binding site can be occupied by activator or repressor
-binding of repressor to enhancer precludes binding of activator to same site
-more likely for activator to remove itself because of it's poor binding affinity



-activators and repressors bind to different DNA sequences
-transcription can be blocked if transcriptional repressor can physically bind to activator thereby inhibiting it


Direct Repression

-direct and physical interactions between repressor and mediator complex
-mediator complex unable to phosphorylate RNA Pol II
-can also exist between repressor and RNA Pol II and/or general transcription factor machinery


Indirect Repression

-repressors can recruit HDAC proteins resulting in removal of acetyl groups from histone proteins
-increases positive charge on core histones tightening grip it has on DNA
-can also recruit chromatin remodeling proteins that twist double helix so CP is inaccessible to general TF's and RNA Pol II



-comprised of opsin protein and retinal chromophore
-responsible for capturing photons and initiating phototransduction cascade
-end result closing of Na channel and difference in voltage across membrane


Opsin Protein

-member of super family of G-protein coupled receptors
-seven transmembrane domains embedded in plasma membrane
-genomes of seeing animals have multiple opsin genes
-different opsin proteins have distinct spectral properties (different rhodopsins capture different wavelengths of light)


Seeing in Color

-photoreceptor neurons must have color sensitive opsins and individual photoreceptors must express only one opsin gene
-non-overlapping pattern of opsin gene expression is used by brian to see world in color


Drosophila Compound Eye

-comprised of ~800 ommatidia
-each ommatidium receives light from 2 degrees of space
-images from each ommatidium integrated within brain and one image is produced


Ommatidium Breakdown

-each contains 8 photoreceptors R1-R8
-photoreceptor neurons homologous to mammalian rod and cone photoreceptors
-each contains rhabdomere containing all phototransduction machinery


Proteins Encoded by Genes Within Fly Genome

-six of them Rh1-Rh6
-Rh1 expressed in photoreceptors R1-R6
-ignore R2
-Rh3 and Rh4 expressed in subsets of R7 cells
-Rh5 and Rh6 expressed in subsets of R8 cells



-absorbs light in orange spectrum
-motion detection


Rh2, Rh3, and Rh4

-absorb light in ultraviolet spectrum


Rh5 and Rh6

-shown that flies can see in color
-absorb blue and green light
-expressed in non-overlapping sets of R8 cells
-70% express Rh5 while remaining 30% express Rh6
-these color sensitive rhodopsin genes restricted to R8 cells


Rh3 and Rh4

-expressed in non-overlapping sets of R7 cells
-70% express Rh3 and remaining 30% express Rh4


R7 vs R8

-if ommatidium contains R7 cells expressing Rh3 then R8 cell will express Rh5
-if ommatidium contains R7 cell expressing Rh4 then R8 cell will express Rh6


Coordination of Rhodopsin Experssion

-single ommatidium can contain one of two pairs of cells (1) Rh3 expressing R7 cell and Rh5 expressing R8 or (2) Rh4 expressing R7 and Rh6 expressing an R8 cell


Properties of Rh5 and Rh6 Enhancer

-review images and example on slide


246 bp Fragment

-upstream Rh6 +1 site sufficient to drive expression of lacZ reporter in pattern of R8 cells identical to that of wild type Rh6 gene
-suggests that all of information required for normal Rh6 expression is contained within this 246 bp element
-determined slightly smaller fragment not only drives expression in R8 cells, but there is some expression in R7 layer
-ectopic expression is result of deleting bases -203 to -246
-suggests that a transcriptional repressor binds to this 42 bp subfragment


Analysis of seq56 Subfragment

-upstream genomic seq of Rh5 and Rh6 genes from 3 different species analyzed for similarities
-42 bases that contain putative site for repressor were analyzed
-analysis of 6 enhancer elements indicated there is a common sequence found in Rh5 and Rh6=seq56
-imperfect palindrome with A and B halves


Experiment with two halves

-wild type 246bp enhancer can drive expression of lacZ in just R8 layer
-mutating B site results in ectopic expression of R7 layer
-mutating A site also leads to ectopic expression of R7 layer
-suggests both halves of seq56 are necessary to prevent expression of Rh5 and Rh6 in R7 cells


Is seq56 sufficient on own to repress transcritpion

-Rh3 gene expressed in subset of R7 and only in R7 layer (with slight exception)
-studies identified enhancer element that can drive expression of lacZ in the normal Rh3 expression pattern
-replace normal control elements with seq56 from Rh6 enhancer abolish expression of lacZ in R7 layer
-now take construct and mutate B half site you restore expression of reporter to R7 cell layer
-similar result seen if mutate A half site
-suggest that seq56 is sufficient to inhibit transcription
-mechanism likely involves binding of repressor to seq56
-repressor likely to be expressed exclusively within R7 photoreceptor neurons


Repressor Prospero

-Rh5 and Rh6 expressed in R7 layer the candidate repressor that binds to seq56 most likely will be expressed exclusively in R7 cell
-search for such TF identified Prospero
-only expressed in R7 photoreceptor
-EMSA assay Prospero shown to bind to seq 56
-mutating B half sites abrogated binding of Prospero to seq 56
-suggests Prospero binds to this site
-mutating A has no effect-indicates it doesn't bind to A only B
-Note: reporter assays both A and B were necessary to repress Rh5 and Rh6, but Prospero only binds to B
-implies second repressor binds to A


Spalt TF

-expressed in both R7 and R8 layer