Exam 3: Lecture 4 Flashcards Preview

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Flashcards in Exam 3: Lecture 4 Deck (17):
1

Dorsal-Ventral Axis (Drosophila) (What?)

-part of embryo that is pattered by Toll pathway.

-Toll receptor expressed uniformly on surface of Drosophila embryo

2

Dorsal-Ventral Axis (Drosophila) (Process?)

-before deposited, remains within female ovary and is surrounded by follicle cells

-ventral follicle cells secrete protein ligand called Spatzle

-all of Toll receptors on ventral surface of embryo bound by Spatzle ligand

-along lateral surface of egg, fewer number of Toll receptors are bound by ligand.

-along dorsal surface of embryo none of receptors are bound by Spatzle

-binding of Toll receptor of Spatzle ligand leads to activation of cytoplasmic signaling cascade

-end result=importation of transcription factor called Dorsal into nucleus

-ventral surface of egg has highest concentration of bound Toll receptors, level of nuclear Dorsal is highest in these nuclei

-cells at lateral regions of embryo will have moderate level of nuclear Dorsal

-all Dorsal protein within cells at dorsal region of embryo located within cytoplasm

3

No Toll Receptor Activation

-without Toll receptor activation, Dorsal protein is held within cytoplasm by Cactus protein

-activation of Toll receptor leads to the degradation of Cactus which allows for Dorsal to be imported into nucleus

4

Dorsal

-is a transcriptional activator -amount of nuclear Dorsal protein leads to differential gene expression along the D/V axis

-of many genes that are directly regulated by Dorsal we will consider three: twist, rhomboid, and short gastrulation

5

Twist

-expression is localized to cells along ventral surface which is area of highest nuclear concentration

6

Rhomboid

-expressed in slightly broader pattern to include a few lateral cells

7

sog

-expressed in a still broader pattern

8

How are twist, rhomboid, and sog expressed in these patterns if all are dependent on Dorsal protein?

-once factor is levels of nuclear Dorsal in ventral and lateral cells

-second concerns the quality of Dorsal binding sites within enhancers of twist, rhomboid, and sog genes

-twist enhancer contains 2 low affinity binding sites

-rhomboid gene contains 3 low affinity and 1 high affinity site -sog gene has 4 high affinity sites, so even at low concentrations of Dorsal protein the sog gene can be transcribed

-low affinity sites within twist means that only cells with high concentrations of Dorsal will express twist -since rhomboid has both types, it will be expressed even in regions of moderate Dorsal levels.

9

Transcriptional target of Dorsal: sna

-snail gene

-sna embryonic enhancer is bound by Dorsal and Twist (a direct target of Dorsal itself)

-both Dorsal and Twist found at high concentrations within nucleus of cells along the ventral side of embryo

-Dorsal and Twist binding sites (within sna enhancer) are low affinity sites

-ensures that sna expression will be limited to cells on ventral surface

10

sna as Transcriptional Repressor

-during later stages of development will bind to short gastrulation embryonic enhancer and will turn its expression off

-contains a zinc finger binding motif

-this domain contains alpha helices that interacts with the major groove of DNA

-represses transcription by binding to C-terminal Binding Protein (CtBP) a transcriptional co-repressor

-CtBP cannot bind to DNA on its own and thus can only exert its effect on transcription by interacting with DNA binding proteins such as Snail.

11

Repressing Transcription Through Regulation of Paused RNA Pol II (Rate of RNA Pol II)

-Drosophila RNA Pol II moves rather slowly only generating transcripts at a rate of approximately 1kb per min

-contrast DNA polymerase adds 1000 bases to the newly synthesized strand every second

-measurements of how long it takes to generate full-length mRNa transcripts have shown that it takes about 2min to fully transcribe a 2kb gene while it takes about 20min before full-length transcripts are seen from a gene that is 20kb in size

-delay (from the time of initiation) correlates well with the in vitro measurements of RNA Pol II rate of transcription

-delay in generating full-length transcripts raises the issue of what happens when a gene needs to be turned off after transcription has been initiated

-RNA Pol II allowed to finish in progress transcriptional elongation or is transcription aborted as soon as transcriptional repressors are recruited to the enhancer/promoter.

-this manuscript from the Levine lab will address this question by examining the mechanism by which the Snail repressor terminates transcription of the short gastrulation (sog) gene in Drosophila.

12

Possible Fates of Elongating Genes after Initiation of Transcriptional Regulation (Graphic Assumes Lagging)

-if assumed complete repression lags behind binding of transcriptional repressors

-in case of small 2kb gene it would be expected that full-length transcripts will be seen for approximately 2 min after transcriptional repressors are first bound to enhancer/promoter elements

-case of large 20kb gene, expectation is that it would take 20 min for last full length transcript to be generated

-times are based on the fact that RNA Pol II, once it has cleared the promoter, moves across the gene at about 1kb per min

-last RNA Pol II molecular to clear the promoter will take 2 min and 20 min respectively to reach the end of a 2kb and 20kb gene

13

Possible Fates of Elongating Genes after Initiation of Transcriptional Regulation (Graphic Assumes No Lagging)

-assumes that transcriptional repressors can very quickly alter the chromatin landscape across an entire gene such that any RNA Pol II molecules that are bound to the gene will be unable to continue with transcriptional elongation

-this model a repression lag is not seen and the production of full-length transcripts cease to be seen very shortly after repressors are recruited to enhancer/promoter elements

-would be the case irrespective of whether the gene is small (2kb) or large (20kb)

14

Transcriptional Repression of sog Gene Involves Lag (Process)

-sog gene contains four exons and three introns

-probes against first and third introns were made and hybridized to Drosophila embryos that are at various stages of development

-if cell is green, first intron is being actively transcribed

-if cell is red, it means last intron is being transcribed (first green intron already spliced out)

-if cell is yellow means some RNA Pol II molecules are transcribing the first intron while other molecules are transcribing the last intron

-in early cell cycle 13 embryos the Snail transcriptional repressor binds to the sog embryonic enhancer and begins to shut off the sog gene

-green signal is seen despite the onset of Snail repression

-in mid and late cell cycle 13 embryos both short and long transcripts are seen (yellow signal)

-since there is a developmental transition between short and long transcripts these results indicate that there is a repression lag

-after each mitosis the memory of repression is erased and the process starts over

-at beginning of cell cycle 14 RNA Pol II is bound to front portion of gene and is transcribing first intron (only green signal)

15

Transcriptional Repression of sog Gene Involves Lag (Results)

-embryos were analyzed for the presence of full length sog transcripts during the entire cell cycle 14 stage of embryogenesis

-in early cc14 embryos RNA Pol II is actively transcribing the 5` end of the gene and thus all cells display a green signal (panels A,G).

-as cells proceed through cc14 most cells display a yellow signal which means that RNA Pol II proteins are bound and transcribing the 5` and 3` ends of the gene (panels B,H).

-at the ventral midline cells being to only display a red signal (panel C,D)

-indicates that no new RNA Pol II molecules are being released from the promoter and that the already bound RNA Pol II molecules are reaching the 3` end of sog.

-near end of cc14 sog transcription is no longer observed -previously transcribed sog transcripts have been spliced and no new transcripts are being made (thus no green or red).

-occurs only the ventral midline because that is where the Snail repressor is expressed.

-remember that Dorsal activates expression of sna at the ventral midline.

-remaining cells within the embryo continue to transcribe the sog gene because the Snail repressor is not present in these cells.

16

Transcriptional Repression of sog Gene Involves Lag (Results)

-embryos were analyzed for the presence of full length sog transcripts during the entire cell cycle 14 stage of embryogenesis

-in early cc14 embryos RNA Pol II is actively transcribing the 5` end of the gene and thus all cells display a green signal (panels A,G).

-as cells proceed through cc14 most cells display a yellow signal which means that RNA Pol II proteins are bound and transcribing the 5` and 3` ends of the gene (panels B,H).

-at the ventral midline cells being to only display a red signal (panel C,D)

-indicates that no new RNA Pol II molecules are being released from the promoter and that the already bound RNA Pol II molecules are reaching the 3` end of sog.

-near end of cc14 sog transcription is no longer observed -previously transcribed sog transcripts have been spliced and no new transcripts are being made (thus no green or red).

-occurs only the ventral midline because that is where the Snail repressor is expressed.

-remember that Dorsal activates expression of sna at the ventral midline.

-remaining cells within the embryo continue to transcribe the sog gene because the Snail repressor is not present in these cells.

17

Transcriptional Repression of sog Gene Involves Lag (Conclusion)

-authors conclude that the Snail repressor is inhibiting the release of paused RNA Pol II.

-based on observations that RNA Pol II has been shown to be stalled between the +30 and +50 site of the sog gene.

-rules out a model in which elongation is inhibited.