Paper Questions

Question 1

• What is being sequenced here?
• What does this figure show?
  
  • What is C? TSS?
• What do the numbers mean?

Answer 1

• What is being sequenced here?
  
  • nascent RNA???
• What does this figure show?
  
  • GRO-seq captures the pieces of RNA that are being generated by active RNA polymerase.
  • ​​This confirms that active promoters and enhancer elements are generally transcribed bi-directionally.
• What is C? TSS?
  
  • The x-axis is the distance from either the enhancer center (C) or the transcription start site (TSS) in kilobases.
• What do the numbers mean?
  
  • ​Number of unique reads

Question 2

Describe the figure

Answer 2

• Gene tracks for the Arid1a gene and enhancer showing ChIP-seq and CLIP-seq data for YY1 cells, as well as GRO-seq reads for mESCs.
• The ChIP-seq analysis in ESCs revealed that YY1 binds to both active enhancers and promoters, with some preference for promoters
• Crick is non-coding strand
• Watson is coding strand

Question 3

Describe the firgures

Answer 3

• YY1 binds to DNA at both enhancer and promoter region with pref. for promotor
• YY1 binds to RNA at both enhancer and promoter region with pref. for promotor.
• YY1 binds with less affinity to enhacners in RNA

Question 4

Describe the figures

Question 5

Describe the figures

Answer 5

• OSN
  
  • Oct4, SOX2, Nanog
  • TF binds to enhancer DNA and not eRNA
• ChIP
  
  • Binding of YY1 to both eDNA and promoter region
• CLIP Crick
  
  • Binding of YY1 to both eRNA and promtor RNA of the non-coding strand
• Clip Watson
  
  • No binding of YY1 to both eRNA and promtor RNA of the coding strand
• Controls
  
  • Only had biotin ligase

Question 6

Describe the figures

Answer 6

Shows the YY1 bindign to both RNA and DNA prmootes gene expression and RNA is affecting the system.

Question 7

Describe the figures

Which experiment(s) are in vivo? Which in vitro?

How does OCT4 compare with YY1 in terms of RNA and DNA binding?

Answer 7

Describe the figures

• Lefty1 DNA is a control from a different gene that Oct4 binds to
• Fig A
  
  • YY1 has preference for promotors over enhancers
  • OCT4 does not bind to RNA
• Fig B
  
  • Competition EMSA to show if OCT4 binds to DNA and not RNA
  • It shows specificity of binding

Which experiment(s) are in vivo? Which in vitro?

• in vitro
  
  • Fig B (EMSA)
• in vivo
  
  • Fig A (CLIP)

How does OCT4 compare with YY1 in terms of RNA and DNA binding?

• RNA->YY1
• DNA-> YY1 and OCT4

Question 8

Describe the figures

Answer 8

• EMSA
• DNA and RNA contained a labled YY1 binding motif
• Increasing [YY1] showed more binding interactions that created more protein-nucleotide complexes
• YY1 binds to both RNA and DNA with preference to DNA

Question 9

YY1 binds to DNA probes containing consensus YY1 binding​ motif in vitro in murine ESC nuclear extracts.

Describe the figures

Answer 9

• EMSA
• RPI30 os a ribosomal protein
  
  • Used to show that YY1 works with more than 1 gene
• When binding motif is removed, no binding occurs .
• When present, the binding motif allows for the protein-DNA complex to be immunopercipitaed out.

FIG B

• 1st lane probe just negative control
• 2nd lane shows that nuclear extract YY1 is binding to probe
• 3rd lane specific competitior is same as the probe but is NOT lableled. Shows that specific competitor binds to YY1 and makes the band disapear.
• 4th lane diferent sequence than the probe that shows that YY1 has sequence specitifity for binding.

Question 10

How do recombinant and endogenous YY1 compare?

Describe these figures

Answer 10

A) Western blot used for invitro studies for later comparison

B

• In vitro competition EMSA between recombient YY1 with competitor DNA
  
  • Lane 1: Negative control
  • Lane 2: YY1 binds to DNA probe
  • Lane 3: Supershifted band when antibody binds to YY1
  • Lane 4: Specific unlabled dna sequence competes for YY1 with probe
  • Lane 5: Non-specific dna sequence does not compete with DNA probe
• Conclusion
  
  • Recombnant YY1 binds to DNA probe in similar ways to endogneous YY1

C) EMSA analysis that is similar to B except with a different gene and RNA competitor.

• Lane 1: Negative control
• Lane 2; recomb. YY1 binds to DNA
• Lane 3: RNA competitor DOES not comepete with DNA for binding
• Conclusion: YY1 binds to DNA and RNA

Question 11

Describe these figures

Answer 11

YY1 binds to RNA at different affinities

Question 12

Different regions in YY1 are responsible for binding to DNA and RNA.

Describe the figures

Answer 12

• These figures show that DNA on Arid1A gene binds to YY1 at YY1’s C terminus, which contains 4 zinc fingers.
• It also shows that it binds to RNA for Arid1A gene binds to YY1 at YY1’s N terminus.

Question 13

Perturbation of RNA levels using transcription inhibitors affects YY1 binding to DNA

Describe the figures

Question 14

Perturbation of RNA levels using transcription inhibitors affects YY1 binding to DNA

Describe the figures

Question 15

Perturbation of RNA levels using transcription inhibitors affects YY1 binding to DNA

Describe these figures

Question 16

Why did the researchers focus on Yin‐Yang 1 (YY1)?

Answer 16

• Yin-Yang 1 (YY1) is
  
  • ubiquitously expressed in mammalian cells
  • plays key roles in normal development
  • and can bind RNA species in vitro

Question 17

What did YY1 bind preferentially: enhancers or promoters?

Answer 17

Promoters

Question 18

What did OCT4 bind preferentially: enhancers or promoters?

Answer 18

Enhancers

Question 19

YY1 sequence motifs were enriched at ______, whereas OCT4 motifs were enriched at ______.

Answer 19

YY1 sequence motifs were enriched at promoters, whereas OCT4 motifs were enriched at enhancers

Question 20

How did they investigate YY1 binding to RNA?

How would you know?

Answer 20

CLIP-Seq because CLIP analyses RNA-protein interactions

Question 21

YY1 binds RNA species at the active _______ and _______ regions where it is bound to DNA.

Answer 21

YY1 binds RNA species at the active enhancer and promoter regions where it is bound to DNA.

Question 22

• At promoters, YY1 preferentially occupied RNA _____ (upstream/downstream) rather than _____ (upstream/downstream) of transcription start sites.

Answer 22

At promoters, YY1 preferentially occupied RNA downstream rather than upstream of transcription start sites.

Question 23

YY1 protein bound both DNA and RNA probes in the ________ assay, with a preference for DNA.

Answer 23

YY1 protein bound both DNA and RNA probes in the EMSA assay, with a preference for ________(DNA/RNA).

Question 24

How does YY1 bind to DNA? to RNA?

What does this suggest about YY1 and how it interacts with DNA/RNA?

Answer 24

• DNA
  
  • Through its 4 zinc fingers in its C-terminal region
• RNA
  
  • Unknown interaction with its N-terminal region
• These results suggest that different regions of YY1 are responsible for binding to DNA and RNA.

Question 25

What was the drybofuranosylbenzimidazole (DRB) treatment used for?

What were the results?

Answer 25

• DRB is a reversible inhibitor of transcription elongation
• They used it to reduce RNA levels at promoters and enhancers without causing changes in the steady-state levels of YY1.
• DRB treatment decreased the transcription of enhancers, promotors, and super-enhancers.
• When DRB was removed, the levels of YY1 increased at promoter and enhancer sites.
  
  • This suggests that nascent RNA produced at promoters and enhancers contributes to YY1 binding to these regions of the DNA.

Question 26

• The exosome reduces the levels of _______ once they are released from Pol II (degradation is 3′ to 5′), so knockdown of an exosome component will cause an _______ in untethered enhancer RNA, which might titrate some YY1 away from enhancers.
• Indeed, exosome knockdown led to _______ steady-state levels of enhancer RNAs and a decrease in the levels of YY1 bound to enhancers
• These results are consistent with the model that YY1 binding to DNA is _______ by binding to nascent RNA.

Answer 26

• The exosome reduces the levels of enhancer RNAs once they are released from Pol II (degradation is 3′ to 5′), so knockdown of an exosome component will cause an increase in untethered enhancer RNA, which might titrate some YY1 away from enhancers.
• Indeed, exosome knockdown led to increased steady-state levels of enhancer RNAs and a decrease in the levels of YY1 bound to enhancers
• These results are consistent with the model that YY1 binding to DNA is stabilized by binding to nascent RNA.

Question 27

What is RPKM?

Answer 27

• When you did RNA-seq, you reported your results in RPKM (Reads Per Kilobase Million)
• These three metrics attempt to normalize for sequencing depth and gene length. Here’s how you do it for RPKM:
  1. Count up the total reads in a sample and divide that number by 1,000,000 – this is our “per million” scaling factor.
  2. Divide the read counts by the “per million” scaling factor. This normalizes for sequencing depth, giving you reads per million (RPM)
  3. Divide the RPM values by the length of the gene, in kilobases. This gives you RPKM.

Question 28

What does ExoKD mean?

Answer 28

Exosome knockdown

• knockdown as opposed to knocking out
  
  • knocking down would be preferenced as knock out would affect much more things in the cell

Question 29

Fig. S12. RNA-seq and Western blot analyses of ESCs, in which an exosome component was targeted with shRNA.

Describe this figure

Answer 29

Question 30

Fig. S12. RNA-seq and Western blot analyses of ESCs, in which an exosome component was targeted with shRNA.

Describe this figure

Answer 30

It implies that (have tanner explain) maybe that RNAi is knocking down what you expect it to knock down

Question 31

RNA‐seq and Western blot analyses of ESCs, in which an exosome component was targeted with shRNA.

Describe these figures

Question 32

What was the point of CRISPR?

Answer 32

Used to bring an RNA close to YY1 without “cutting”

Question 33

Describe this figure

Question 34

How did they test the idea that RNA near regulatory elements can contribute to stable TF occupancy in vivo?

Answer 34

• They tethered RNA in the vicinity of YY1 binding sites at six different enhancers in ESCs using the CRISPR/Cas9 system and determined whether the tethered RNA increases the occupancy of YY1 at these enhancers.

Question 35

What happened in the CRISPR experiment?

Answer 35

• Tethering the Arid1a RNA at each enhancer led to increased binding of YY1 to the targeted enhancer as measured by ChIP-qPCR
• This elevation in YY1 binding was specific to the targeted locus and the sequence of tethered RNA as there was no observable increase in YY1 binding at the enhancers not targeted in the same cells (Fig. 4B) or targeted with tethered RNA not compatible with YY1 binding in vitro (fig. S13).
• These results show that RNA tethered near regulatory elements in vivo can enhance the level of YY1 occupancy at these elements.

Question 36

Describe these Figures

Question 37

Describe these figures

Question 38

Does tethered RNA increase YY1 binding affinity?

Describe these figures

Question 39

Does tethered RNA increase YY1 binding affinity?

Describe these figures

Question 40

What do these bitch ass figures show?

Question 41

Different regions in YY1 are responsible for Fig S8 binding to DNA and RNA. (Fig S8)

Describe the figures

Question 42

What the fuck is going on this diagram?

Question 43

Describe this figure