L7: The Genome Strikes Back

Question 1

What can counter-act the previously mentioned TEs?

Answer 1

Epigenetic marks such as tri-methylation counteract these repetitive elements

Question 2

How did Jacobs and other research investigate the repression of these repetitive elements?

Answer 2

Investigating the most impopular DNA sequences in our genome: KRAB Finger genes. There are tonnes of these in the genome and researchers often discarded them and found them annoying more than interesting

Question 3

What characteristic traits of Zinc fingers likens them to TEs? Where are they located?

Answer 3

Zinc fingers were once themselves very repetitive and are located near the centromeres & telomeres

Question 4

Zinc finger genes.. What kind of creatures are they?

Answer 4

KRAB Zinc finger genes are transcriptional repressors. One of the oldest forms of TF, they are really a DNA binding domain, their structure in a series of hoops each designed to bind to specific nucleotides and thus their structure alligns with specific nucleotides. Each of these loops are known as a finger and each protein can have between 10-35; each finger interacts with one amino acid sequence of 3 nucleotides; therefore the sequence of the fingers determines their DNA binding sequence

Question 5

How were zinc fingers previously used in research?

Answer 5

Could design a Zinc finger attached to a repressive domain to repress a particular (cancerous) gene; CRISPR killed this booming field as guiding RNAs are much easier to design than proteins (hard to figure out folding etc)

Question 6

What trend has been seen in KRAB ZFs in evolutionary history?

Answer 6

KRAB-ZNF clusters have greatly expanded in higher vertebrates and primates in particular. Often in unstable regions as these are difficult to replicate perfectly; These then duplicate frequently and begin very similar but evolve rapidly, this causes differences frequently in their ZFs aka their DNA binding domains, zinc fingers downstream or upstream can affect this new duplication. KRAB ZNF regions are therefore loaded with signals of positive selection

Question 7

Describe the lineage of KRAB ZFs in primate evolution

Answer 7

400 in total and ~170 primate-specific KRAB- zinc finger genes in the human genome. 23 ZFs were present in the split of primates from non-primates, 67 in the simian split, 66 in the catarrhine split and 14 in the hominid split. These are also likely polymorphic within species

Question 8

Do mice have KRAB ZFs?

Answer 8

Yes, a mouse-specific KRAB zinc finger protein ZFP809 protects mouse ESCs against retroviral DNA insertions in embryonic stem cells

Question 9

What role could Zinc Fingers therefore play in regards to retrotransposons?

Answer 9

Genome’s defense mechanism against retrotransposon invasions

Question 10

What question and therefore hypothesis did Jacobs propose to a class in 2009?

Answer 10

QUESTION
Is there a link between the dramatic expansion of KRAB zinc finger gene clusters in primates and episodes of retroviral attacks on primate genomes

HYPOTHESIS
Invasion of primate genomes by retrotransposons elicits a ‘Host- response’ to deal with unwanted endogenous retroviral activity.

The vast expansion of KRAB ZNF genes in primate genomes equips the host’s genome with the tools to response to newly emerged retrotransposons during primate evolution

Question 11

How do KRAB ZFs exert their repressive actions?

Answer 11

KRAB ZNFs recruit ‘KRAB domain associated Protein 1’ (KAP1) to exert their repressive actions: Knock Out of Kap1 in mouse ESCs results in re-activation of endogenous retroviral (ERV) gene expression. KAP1 and KRAB ZNFs keep a lot of unwanted ERV viral elements silenced

Question 12

How did Jacobs test where these KRAB ZFs bound in the genome?

Answer 12

Had meh antibodies for these ZFs and wanted to use use CH-IP seq to see where they bind, so instead since they had good antibodies for KAP1 to see where these bound, as they believed they worked together for the silencing

Question 13

Do KRAB ZFs suppress human specific TEs?

Answer 13

Yes, Primate-specific transposable elements are bound by KAP1 in human embryonic stem cells. SVAs emerged late; around 10m years ago. KAP1 seems to preferentially mark these primate specific and late emerging regions in our genome (Also biased towards late emerging LTRs, L1HS, L1PA)

Question 14

Are KAP1s more primate specific then?

Answer 14

No, they are specific to the species; KAP1 binds to MOUSE-specific retrotransposons in mouse embryonic stem cells (Mostly IAPs)

Question 15

In the meantime, what was being observed about the evolution of ZFs in 2011?

Answer 15

There was a trend suggesting a trend between retroelements and tandem zinc finger genes suggesting a coevolution

Question 16

What hypothesis did Jacobs draw from this?

Answer 16

Are KRAB ZNFs part of a species-specific defense mechanism against retrotransposon invasions?

Question 17

How did Jacobs first set about to prove that KRAB ZNFs part of a species-specific defense mechanism against retrotransposon invasions

Answer 17

Its hard to prove this, but during journal club saw a DS model in which they added a human extra chromosome 21 and after some time realised this could be a good model system.

To test this, they determined the fate of primate-specific retrotransposons in a non-primate background using trans-chromosomic mESCs that contain a copy of human chromosome 11 (E14(hChr11) cell.

Question 18

What were the findings of the trans-chromosomic mESC paradigm?

Answer 18

In the TC11-mESC cellular environment, primate-specific retrotransposons, including SVA and L1PA elements, are derepressed and gain activating histone H3 Lys 4 (H3K4me3) marks.

As a result of this de-repression, a majority of SVA (51%), human-specific L1 (L1Hs) (93%) and some other L1PA elements, such as L1PA4 (16%), become aberrantly transcribed. These findings suggest primate-specific retrotransposons have a transcriptional potential that is repressed by primate-specific factors.

Question 19

What were the ‘promising candidates’ for these primate specific factors repressing transposons?

Answer 19

Promising candidates for these factors are the approximately 170 KZNF genes that emerged during primate evolution.

Question 20

From these 170 primate specific ZFs, how did they select their ‘prime suspects’?

Answer 20

They reasoned that a KZNF gene responsible for protecting genome integrity, most critical in the germ line, must be highly expressed in hESCs. So they focused on 14 highly expressed, primate-specific KZNF genes. Some picked up by RNAseq, some were synthesised as they were so repetitive they were hard to pick up from the RNA

Question 21

Once they had selected their 14 prime suspects, how did they go about testing them?

Answer 21

They tested each candidate for a role in repressing SVA retrotransposons, which first appeared in great apes 18–25 million years (Myr) ago, and are still active.

They set up a luciferase assay based screen in mESCs in which an SVA element cloned upstream of a minimal SV40 promoter strongly enhances luciferase activity. Each candidate KZNF was co-expressed with the SVA–luciferase construct to determine its effect on reporter activity.

Question 22

What was the results of the KRAB-SVA luciferase reporter study in regards to further selection from the prime suspects?

Answer 22

Of all KZNFs tested, ZNF91 most dramatically decreased SVA-driven luciferase activity, reducing activity to 16 ± 4% relative to an empty-vector-transfected control.

Some other KZNFs had modest effects on this reporter, but were not further analysed, as those with the strongest effect also inhibited the OCT4 (also known as POU5F1) enhancer, which is not KAP1-bound in ESCs, and therefore suggests a nonspecific effect

Question 23

What did they find regarding the structure of the SVA and its repression by KZNFs?

Answer 23

Structure–function analysis of SVA revealed that the variable number tandem repeat (VNTR) domain is necessary and sufficient for ZNF91-mediated repression of luciferase activity.

Question 24

How were the findings regarding KZNF repression linked back to the TC11-mESCs?

Answer 24

Transfection of TC11-mESCs with human ZNF91 restored the repression of deregulated SVAs on human chromosome 11, causing a strong decrease of the aberrant H3K4me3 ChIP-seq signal at SVAs, while leaving other derepressed elements such as L1Hs or L1PAs unaffected.

Transfection of ZNF91 also significantly repressed aberrant transcription of SVA repeats, indicating that ZNF91 is sufficient to restore transcriptional silencing of SVAs.

Question 25

Was ZNF91 alone in its effects on SVAs, did other KZNFs also repress them?

Answer 25

No such effects were observed for other primate KZNFs (ZNF90, ZNF93, ZNF486, ZNF826, ZNF443, ZNF544 or ZNF519) transfected in TC11-mESCs, validating the specificity of the ZNF91–SVA interaction

Question 26

How specific was KZNF91 to SVAs? What theoretical implication does this have?

Answer 26

Cellular genes near SVAs on human chromosome 11 in TC11-mESCs were also repressed by ZFN91, with the distance of a gene to an SVA as the major factor governing the amount of bystander repression, supporting the hypothesis that the host response to retrotransposon insertion has significantly impacted human gene expression patterns

Question 27

When did ZNF91 emerge and has it changed since?

Answer 27

ZNF91 emerged in the last common ancestor (LCA) of humans and Old-World monkeys and has undergone dramatic structural changes, including the addition of seven zinc-fingers in the LCA of humans and gorillas.

Question 28

How did Jacobs explore the LCA of ZNF91?

Answer 28

They reconstructed ancestral versions of ZNF91 by parsimony analysis and found that ZNF91 as it probably existed in the LCA of humans and gorillas (ZNF91hominine) was able to repress the SVA-luciferase reporter in a similar fashion to human ZNF91. However, ZNF91 as it existed in the LCA of humans and orangutans (ZNF91great ape) only reduced luciferase activity to around 80% of baseline and macaque ZNF91 completely lacked the ability to repress SVA-driven luciferase activity.

Question 29

How was the importance of the seven recently added hominine Zinc Fingers further analysed? What was concluded from this line of research?

Answer 29

The importance of the seven recently added hominine zinc-fingers was further supported by deletion analysis of ZNF91. These findings suggest that the changes in ZNF91 between 8–12 Myr ago have markedly improved the protein’s ability to bind and repress SVA.

Question 30

What interesting finding did Jacobs first observe regarding L1H elements which encouraged him to probe further?

Answer 30

In our KAP1 ChIP experiments, KAP1 also showed a strong association with the 5′ untranslated region (UTR) of L1PA elements. None of the 14 KZNFs had a significant effect on the 5′ UTR of the current active L1Hs cloned upstream of the luciferase reporter when tested in mESCs.

Question 31

Did these KZNFs have any effects on LINE elements then?

Answer 31

Yes, ZNF93 significantly reduced luciferase activity of a reporter with the 5′ UTR of a KAP1-positive L1PA4 element.

Question 32

How did Jacobs verify the recruitment of ZNF93 to L1PA4 elements on the human genome? What did they find?

Answer 32

They performed ChIP-seq analysis on hESCs using antibody ab104878, which recognizes ZNF93 and co-immunoprecipitates KAP1. They found that ZNF93 binds to the 5′ end of L1PA4, the ancestral subtypes L1PA6 and L1PA5, and the descendant subtype L1PA3. No consistent ZNF93 binding was detected at L1PA7 or older subtypes nor at the most recently evolved L1PA2 and L1Hs.

Question 33

How can the lack of consistent binding to L1PA2 and L1Hs be explained?

Answer 33

Comparative sequence analysis revealed that the absence of ZNF93 binding in L1Hs and L1PA2 can be explained by a 129-base-pair (bp) deletion in the 5′ UTR that spans the ChIP-determined ZNF93- and KAP1-binding sites. The deletion is also present in ∼50% of L1PA3 elements, resulting in distinct subgroups of shorter (L1PA3-6030) and longer (L1PA3-6160) L1PA3 elements, but is not present in L1PA4–6 families.

Question 34

How did Jacobs investigate the interaction of ZNF93 with the 129-bp L1PA element?

Answer 34

To investigate the interaction of ZNF93 with the 129-bp L1PA element, they tested a series of L1PA4 segments cloned upstream of an OCT4-enhancer fused to an SV40-promoter and luciferase-reporter in mESCs. Both the 129-bp element and a 51-bp sub-fragment were sufficient to confer ZNF93-mediated repression of the luciferase reporter, and this repression was abolished by elimination of the 51-bp portion in the 129-bp fragment

Question 35

How did these findings regarding the 51bp segment correspond to in-silico models?

Answer 35

The 51-bp element encompasses a computationally predicted DNA binding motif for the 17 fingers of ZNF9320 and the central 18 bp of this region displays strong similarity to the predicted recognition motif of zinc-fingers 8–13 of human ZNF93.

Question 36

How was this KZF93 in-silico prediction validated?

Answer 36

A ZNF93 variant that has all contact residues in zinc-fingers 8–13 replaced by serine residues (ZNF93serF), a modification that abolishes DNA binding selectivity, was unable to repress luciferase activity of the L1PA4 elements, suggesting that fingers 8–13 of ZNF93 are important for recognition of the 129-bp element in L1PA3-6 retrotransposons.

Question 37

When did KZNF93 emerge and how has it changed?

Answer 37

ZNF93 emerged in the LCA of apes and Old-World monkeys and reconstruction of the evolutionary history of the ZNF93 protein by parsimony suggests that dramatic changes took place in the LCA of orangutans and humans between 12–18 Myr ago (ZNF93great ape).

Question 38

How did Jacobs further explore the LCA of KZNF93?

Answer 38

Indeed, macaque ZNF93 does not have the ability to repress the 129-bp or 51-bp element of L1PA4 in the luciferase assay, but ZNF93great ape represses at levels similar to ZNF93human, suggesting changes in the ape lineage probably enabled ZNF93 to regulate L1 activity.

Question 39

How did Jacobs further explore this 129bp deletion?

Answer 39

To explore the function of the lost 129-bp element, we created a version of L1Hs with this sequence restored in its 5′ UTR (L1Hs+129L1PA4), or a scrambled version of this 129-bp sequence (L1Hs+129scrambleL1PA4) as a control, and compared retrotransposition efficiencies to wild-type L1Hs in HEK293FT cells in an in vitro retrotransposition assay. In this assay, a retrotransposition event results in green fluorescent protein (GFP) expression (intron in middle of GFP domain- splicing completes domain).

L1Hs+129L1PA4 shows a 1.76-fold (± 0.45 s.e.m.) higher retrotransposition activity compared to wild-type L1Hs, an effect not seen with L1Hs+129scrambleL1PA4, suggesting that this 129-bp sequence promotes retrotransposition.

Importantly, co-expression of ZNF93 significantly reduced retrotransposition of L1Hs+129L1PA4 to just 24% (± 3% s.e.m.) relative to L1Hs, but had no significant effect on L1Hs+129scrambleL1PA4

Question 40

What conclusions did Jacobs draw from this research on KZNF93 and LINE elements?

Answer 40

These data suggest the 129-bp sequence, as it once existed in the 5′ UTR of L1PA subfamilies, may have been beneficial to L1 mobilisation, but since ZNF93 evolved to bind this element, losing it allowed the L1 lineage to escape ZNF93-mediated repression, providing net selective advantage

Question 41

How did phylogenetic analysis of L1PA3 elements provide further evidence for this conclusion regarding the 129bp in LINE elements?

Answer 41

Phylogenetic analysis of L1PA3 elements and calculation of the average distance of L1PA3-6030 and L1PA3-6160 elements from the respective consensus sequences, suggests that L1PA3-6030 elements lacking the 129-bp element have expanded more recently in our genome than L1PA3-6160 elements, showing an estimated age of 12.5 and 15.8 million years. This strongly suggests that loss of the ZNF93-binding site—and thereby the evasion of the host repression—propagated a new wave of L1 insertions in great ape genomes.

Question 42

How are these findings regarding SVAs and KZNFs reflected in other apes?

Answer 42

In a similar fashion, the structural changes in ZNF91 allowing it to repress SVA elements may have driven the further evolution of new and different SVA-subtypes in gorillas, chimpanzees and humans, a pattern that is not observed in orangutans, which diverged before ZNF91 had undergone these structural changes. Notably, the size of the VNTR region of SVA, the prime interaction site of ZNF91, has increased during the timeframe of structural changes to ZNF91

Question 43

Collectively, describe the model supported by the data collected by Frank Jacobs in this study?

Answer 43

Our data support a model in which modifications to lineage-specific KZNF genes are used by the host to repress new families of retrotransposons as they emerge, which in turn drives the evolution of newer families of retrotransposons, in a continuing arms race. Because repression affects nearby genes, KZNFs have probably been co-opted for other functions that persisted long after the original transposon expansion they first evolved to repress had subsided, fuelling the evolution of more complex gene-regulatory networks.

Unlike an arms race with an external pathogen, retrotransposons are host DNA, suggesting that a mammalian genome is itself in an internal arms race with its own DNA, and thereby inexorably driven towards greater complexity.

Question 44

What did a further study by Imbeault et al show in 2017?

Answer 44

They amplified all of these KZNFs and showed that likely multiple zinc fingers work to repress the same site, some selected to do this exact job.

They found clear support for the arms race model at the 5′ ends of primate-specific L1 elements (L1PA), where ZNF141, ZNF649, ZNF765 and the previously described ZNF93 were found to be dynamically linked with their targets in recent evolution, with zinc-finger mutations accumulating coincidentally with the appearance of new L1PA subfamilies, and loss of binding sites for all of these KZFPs in the newest human-specific L1 (L1Hs) elements.

They could retrace specific mutation events in the binding motifs of KZFPs that correlated with loss of binding in the youngest elements, and these events were subtler than the 129-bp deletion event that led to escape from ZNF93.

Question 45

What are some further consequences of the evolutionary armsrace between KZNFs and TEs?

Answer 45

Adaptive evolution of gene promoters are seen to be driven by primate-specific KRAB zinc finger proteins. They bind to gene sequences nothing to do with TEs

This is explained by the promoters being very similar to these TEs; by chance it might start to recognise promoters.

Similar to viruses, if you target these elements you might accidentally start to target the promoters that these viruses originally evolved to repress

Question 46

How may KZNFs’ function evolved past supression of TEs? Does this align with the evidence?

Answer 46

In theory, it is possible that after the KZNFs became redundant for TE control, its binding specificity changed under pressure of some of the genes it evolved to regulate as part of its co-opted function. This does not seem to be the case however: A multiple sequence alignment of the protein sequences shows very few differences in the zinc finger (ZNF) domains and contact residues of all three KZNFs between the oldest lineages and humans

Question 46

What evolution is observed as a result of KZNFs binding to promoters?

Answer 46

Most KZFNs measured show a pattern of reduction in conservation at the binding site of the promoter-bound KZNF compared to the flanking sequences up and downstream. Therefore there seems to be an increase in evolution around these sites.

Question 47

Why might there be this increase in evolution around these sites?

Answer 47

Could either be that the ZF induces this or the have a tendency to bind to evolving sequences. It does, however, support their hypothesis that the emergence of KZNFs can exert selective pressure on the KZNF binding sites in gene promoters. This could possibly be to restore the imbalance caused by the repression of the KZNFs.