Successfully Reported ChIP Cloning for New Target Identification

The first demonstration that formaldehyde crosslinking combined with the modified chromatin immunoprecipitation assay can be used to clone promoters that are direct in vivo targets of a mammalian TF was published by Weinmann et al. in 2001 [19]. They cloned, confirmed, and described three novel E2F target genes. So far, subsequent studies have described new targets for E2A [23], Egr1 [24], EWS/ATF-1 [25], Drosophila TF engrailed [26], RUNX1 [20], BARX2 [21], Smad4 [22]. These studies are summarized in Table 7.1. Solana et al. [26] were the only group to use UV light instead of formaldehyde for crosslinking Drosophila embryos before isolating geno-mic targets. Some studies varied the conventional cloning after immunoprecipita-tion. For example, Jishage et al. [25] used the transcriptional activity of the fragments for detection (see Section 7.4.2), and DeBelle et al. [24] used a multiplex PCR step for target identification. The literature summary shows that some confirmed targets failed to contain consensus binding sites [19, 26] (for discussion see Sections 7.2.6 and 7.4.1). Unfortunately, only a few of the predicted consensus sites have been tested by EMSA. The ratio of confirmed targets to total reported clones varied among the studies but nevertheless show that the ChIP cloning strategy was normally limited to the identification of a small number of target genes. Multiple targets are localized (if actual information is available) in intronic regions [21, 23, 26]. Greenbaum et al. [23] were able to confirm immunoprecipitation of the putative promoter region

Tab. 7.1 Summary of reports of successful ChIP cloning for new target identification with subsequent validation.

Reference

Crosslink

TF

Species

Total clone number reported

Number of confirmed targets

Localization

With consensus site

EMSA a|

Reporter gene assay

Cene expression monitoring

19

FAb>

E2F

human

28

9

3 promoter 6 no information

1 out of 3 analyzed

2 out of 2 analyzed

2 out of 2 analyzed

3 out of 3 analyzed

23

FA

E2A

mouse

13

8

1 intronic sequence 7 no information

8

no

no

1 out of 1 analyzed

24

FA

Egrl

human

1

1

no information

no information

1 out of 1 analyzed

1 out of 1 analyzed

1 out of 1 analyzed

25

FA

EWS/ATF-1

human

62

6 out of 16 clones with consensus site

3 clones in the vicinity or inside of genes

3 clones far away from transcription initiation site

6

1 out of 1 analyzed

6 out of 6 analyzed

6 out of 6 analyzed

26

UV light

engrailed

Drosophib

542

no ChIP confirmation, 203 further analyzed

47% intronic 53% intergenic

49 out of 107

4 out of 4 analyzed

1 out of 1 analyzed

12 out of 14 analyzed

20

FA

RUNX1

human

1

1

promoter

1

1 out of 1 analyzed

1 out of 1 analyzed

1 out of 1 analyzed

21

FA

BARX2

human

60

21

25 % intronic 35% within 50 kb up or down of annotated genes

30 % greater than 50 kb from a gene

13 sites reported

9 out of 13 analyzed

14 out of 19 analyzed

8 out of 11 reduced by RNAi

22

FA

Smad4

mouse

60

1

promoter

yes

no

1 out of 4 analyzed

1 out of 1 analyzed

a) Electrophoretic mobility shift assay.

b) Formaldehyde.

of a newly identified intronic E2A target clone, suggesting multiple binding sites for the factor. Intronic enhancers have become well known in recent years [51] and are located predominantly in intron 1 and intron 2. For example, intron enhancers have been described for three HNF4a targets (aldolase B [52, 53], apolipoprotein B [54], and adenosine deaminase [55]).

Some publications have reported similar or modified approaches to isolating genomic fragments, but those studies did not use follow-up experiments to confirm in vivo binding of the factor of interest to the isolated DNA [38, 56-63]. One of these studies used a UV laser instead of formaldehyde for crosslinking [38]. But, due to the lack of in vivo confirmation, it is difficult to be sure if any of the clones in these studies corresponded to real in vivo targets [18].

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