Whyshouldwelookatthegenomein3D

!BMC,Nov27th2015lvaroMartnezBarrio,PhDAlvaro.Martinez.Barrio@scilifelab.selinkedin.com/in/ambarrio@ambarrio

-log 1

0P

-log 1

0P

AB1NS*

Atlantic Ocean

SBSkagerrak

BH*BSBV

Baltic Sea

0.08

BAS30

BAH60

BAS16

NS56

BAS39

BAH30

SB40

BAV33

SB14

NS8

BAH51

NS34

SB50

NS7

BAH39

BAH8

NS57

BAH53

BAS55

BAH5

AB4

SB20

SB5

AB56

BAS1

BAS21

BAV58

BAH10

NS13

BAV38

AB25

BAS7

SB41

BAH19

BAH59

SB30

BAH43

NS22

AB29

BAS28

SB16

AB28

NS43

SB44

SB48

SB18

SB38

AB10

AB31

BAV47

NS54

SB60

NS44

SB33

AB18

BAS35

BAV6

BAS43

SB4

BAV22

SB55

NS47

BAH13

SB47

BAH3

BAH42

BAH28

AB6

NS46

SB2

AB26

BAS47

SB22

BAH32

AB49

BAH14

AB48

NS39

BAS14

NS2

BAV10

AB50

NS15

BAS3

NS53

BAV12

BAH52

NS1AB1

BAH35

BAH33

BAH26

AB13 NS49

NS5

BAS6

BAS20

BAH54

BAH48

BAH56

BAS36

BAV18

SB27

NS17

BAV28

BAH38

BAS40

NS3

AB35

AB9

BAS49

SB42

BAH22

AB8

BAV14

BAS45

BAS53

BAH2

BAH12

NS42

BAV27

NS9

NS19

BAS37

NS37

BAS25

SB28

AB14

BAS58

NS59

BAS46

NS30

AB45

BAV4

BAH11

NS55

AB2

AB47NS52

BAS60

BAS52

SB54

BAV16

AB19

NS32

NS45

BAV34

BAS38

BAH45

BAS11

SB29

SB1

SB13

AB43

AB11

NS12

BAS10

NS40

NS33

SB19

NS16

BAV40

BAS54

SB26

BAH57

BAV56

BAH29

BAS56

BAS5

BAH18BAV43

NS14

BAH44

SB15

BAV37

SB8

NS27

BAV45

BAV36

BAS32

NS41

BAS34

BAV55

BAH37

AB42

AB55

BAH24

SB37

BAV8

BAH55

BAS4

BAV24

SB56

NS50

BAV30

NS35

BAV17

SB3

NS60

NS24

AB51

NS6

SB43

SB12

NS23

BAH17

NS38

NS11

BAV49

AB34

BAV52

BAH23

BAS19BAS27

AB40

SB45SB11

BAH47

SB53

NS48

BAH4

BAV59

AB21

BAS33

AB38AB20

BAV48

BAV9

SB31

BAV2

BAH21

BAH36

BAV29BAV35

BAH20

BAV11

NS25

NS21

BAS9

SB52SB10

SB9

NS26

BAV26

NS10

BAH46

BAS57

SB17

SB25

BAV32

BAS41

AB59

NS31

AB30

BAH9BAH49

AB54

SB49

BAV1

AB27

BAV5

BAS42

BAV39

AB22

NS51

BAV50

AB12

AB32

AB39

SB34

AB41

BAV15

BAS15

SB6

AB24

BAV53

SB35

AB60

BAS13

AB44AB57

BAS18

BAS50

BAV13

BAV54

AB15

AB3

BAS17BAV23

SB59

BAV51

BAH41

AB46

SB58

BAS22

BAH27

BAS24

BAV3

NS36

BAS51

NS28

BAH34

AB36

BAH7

SB36

SB21

BAS48

AB17

BAS12

BAV57

NS58

AB16

BAV41

BAH40

AB7

AB37

BAH25

NS20

SB24

BAV19

BAV20

BAH31

BAV21

BAS8

NS18

BAV60

BAV44SB46

BAS26

AB33

SB7

SB23

BAV42

AB53

SB57

BAS2

BAV25

AB58

BAH58

SB32

BAH6

AB52

NS29

BAV31

BAH16

BAH15

BAS23

BAH50

BAS29

BAS44

BAV46

BAS59

SB51

AB23

AB5

BAS31

BAV7

SB39

NS4

A

s218

s1523

s2123

s273s899

0

50

100

150

200

250

300

-log

10

(P)

SNP position

s346

0.05

.

NS*AB1

AI

SBSH

BF*

BH*KB

KTBA

BRBK

BUBSBV

BHBGBVBC

PH

B

D

Salin

ity(

)

1.836 Mb 1.842 Mb

Normalized copy number

2 10020 40 60 80

1 8361 8361 836 MbMbMb

3

6

6

7

7

12

20

25

35

35

Pop

s

AI

AB1

NS*

SH

SB

KB

KT

BF*

BC

BR

BA

BG

BV

BH

BH*

BS

BV

BU

BK

PH

7

6

6

6

6

8

23

25

35

35

High choriolytic enzyme 2

Atlantic Ocean

C

FBX

W7

FHD

C1

AR

FIP

1

ND

UFA

F2

TMEM

2

PG

F5

FOX

D5

NR

N1

PR

LR

HFE

MH

C-I

LRR

C8

C

RR

EB1

AB1NS*

BH*BSBV

s218119.4 kb

s152333.58 kb

s89910.93 kb

s212366.51 kb

s27332.66 kb

NR

N1

s152333.58 kb

PR

LRs89910.93 kb

FBX

W7

FHD

C1

AR

FIP

1

ND

UFA

F2

TMEM

2

PG

F5

FOX

D5

s218119.4 kb

HFE

MH

C-I

LRR

C8

C

s212366.51 kb

RR

EB1

s27332.66 kb

Baltic Sea

SkagerrakSB

-log

10

(P)

50

100

0

150

200

00.20.40.6

0.81

Fst

119.4 kb

scaffold331

-log

10

(P)

Gap

E

KLHL33 SLC12A3CBLN3 KLHL33

C1QL4

Gap

~ 65 kb

Fig. 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0

Alle

le f

req

uen

cy

Figure 2 Genetic differentiation between Atlantic and Baltic herring. (A) Manhattan plotof significance values testing for allele frequency differences between pools of herring frommarine waters (Kattegat, Skagerrak, Atlantic Ocean) versus the brackish Baltic Sea. Lowerpanel, corresponding plot for scaffold 218 only; both P- and FST - values are shown. (B)Neighbor-joining phylogenetic tree based on all SNPs showing genetic differentiation in thiscomparison (P < 10-20). (C) Comparison of allele frequencies in five strongly differentiatedregions. The major allele in the AB1 sample (Atlantic Ocean) was used as reference at eachSNP. Lower panel, neighbor-joining tree based on haplotypes formed by 128 differentiatedSNPs from scaffold 218. (D) Heat map showing copy number variation partially overlappingthe HCE gene. Orientation of transcription is marked with an arrow. Population samples andsalinity at sampling locations are indicated to the right; abbreviations are explained in Table 2. (E) Strong genetic differentiation between Atlantic and Baltic herring in a region downstream of SLC12A3; statistical significance based on the 2 test is indicated.

-log 1

0P

Whyshouldwelookatthegenomein3D

http://webvideomarketingportugal.com/httpthenextweb-commedia20130920the-future-of-cinemas/

http://webvideomarketingportugal.com/httpthenextweb-commedia20130920the-future-of-cinemas/

-log 1

0P

WhereismycausativeSNP? Candidategeneapproach(100kbwindows) Inferpathwaysthatarecommon

12

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

13

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

14

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

15

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

16

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Stormo,G.D.etal.NucleicAcidsResearch(1982)

17

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Stormo,G.D.etal.NucleicAcidsResearch(1982)

ChIP-exoAn extension of chromatin immunoprecipitation followed by sequencing (ChIPseq) that includes exonuclease trimming after ChIP to increase the resolution of the mapped transcription factor bound sites.

regulating transcription to establish cell-lineage-specific programmes. Experiments usingthe ChIP-exo technique uncovered a 52-bp CTCF-binding motif that contains four CTCF-binding modules15,16 (FIG.1).

The presence of CpGs in the DNA consensus sequence of the CTCF-binding site supports the notion that methylation of cytosine residues at carbon 5 of the base to form 5-methylcytosine (5mC) in CpG-containing sites may, at least partly, underlie CTCF tar-get selectivity in different cell types17. Recent studies indicate that DNA methylation has a widespread role in regulating CTCF occupancy at many genes, includ-ing CDKN2A (which encodes INK4A and ARF)18, B-cell CLL/lymphoma 6 (BCL6)19 and brain-derived neurotrophic factor (BDNF)20. One study has mapped the occupancy of CTCF in 19 human cell types; by comparing this information with DNA methylation data from parallel reduced representation bisulphite sequencing, it was found that 41% of cell-type-specific CTCF-binding sites are linked to differential DNA methylation21 (FIG.2). Conversely, at 67% of sites that showed variability in DNA methylation, the presence of 5mC was associated with a concomitant downregula-tion of cell-type-specific CTCF occupancy. CTCF can also affect the methylation status of DNA by forming a complex with poly(ADP-ribose) polymerase 1 (PARP1) and DNA (cytosine-5)-methyltransferase 1 (DNMT1). CTCF activates PARP1, which can then inacti-vate DNMT1 by poly(ADP-ribosyl)ation, and thus

maintains methyl-free CpGs in the DNA22,23. An addi-tional level of complexity in the interaction between CTCF and its target sequence can arise from the oxida-tion of 5mC to 5-hydroxymethylcytosine (5hmC)24,25, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC)26 by ten-eleven translocation (TET) enzymes. Genome-wide profiling analyses of 5hmC have shown that this modification and, to a lesser extent, 5fC are enriched genomic locations that contain CTCF-binding sites27,28. Furthermore, identification of proteins that bind to different oxidized derivatives of 5mC discov-ered CTCF as a 5caC-specific binder29. These results underscore the complexity and possible importance of the relationship between DNA methylation status and plasticity of CTCF occupancy. However, the presence of cell-type-specific CTCF-binding sites that are not differentially methylated suggests the existence of other mechanisms by which the DNA occupancy of this protein is regulated (FIG.2).

One such mechanism is post-translational cova-lent modification of CTCF, such as sumolyation30 and poly(ADP-ribosy)lation31. In breast cancer cells, defec-tive poly(ADP-ribosyl)ation of CTCF leads to its dissoci-ation from the CDKN2A locus, which results in aberrant silencing of this tumour suppressor gene32. In Drosophila melanogaster, poly(ADP-ribosy)lation of Centrosomal protein 190 kDa (Cp190) and CTCF facilitates their interaction, tethering to the nuclear matrix and intrachromosomal contacts33.

Figure 1 | Features of CTCF-binding sites in the genome. Binding sites of CCCTC-binding factor (CTCF) are associated with different genetic elements. The majority of these sites are intergenic and colocalize with cohesin. In

genes and short interspersed nuclear elements (SINEs)) and extra-TFIIIC (ETC) loci, which suggests that TFIIIC and CTCF

cooperate in some nuclear processes. The 12-bp consensus sequence of CTCF-binding sites is embedded within binding

modules 2 and 3 as determined by ChIP-exo experiments. DNA methylation (represented by red circles) of cytosine

residues occurs at positions 2 and 12 of the consensus sequence in a subset of CTCF-binding sites.

Nature Reviews | Genetics

TFIIIC

ETC locus

B box

Promoter

Cohesin

Pol III

Enhancer

Gene

Condensin

tRNA geneand SINE

CTCFCTCF

CTCF CTCF

Module 2Module 1 Module 3 Module 4

CTCF

ETC locus

REVIEWS

NATURE REVIEWS | GENETICS VOLUME 15 | APRIL 2014 | 235

2014 Macmillan Publishers Limited. All rights reserved

OngC-tandCorcesV.G.NatureReviewGenetics2014

18

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

19

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Lindblad-Toh,K.etal.Nature(2011)

20

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Kim,T.H.andB.Ren,AnnuRevGenomicsHumGenet,2006

21

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Kim,T.H.andB.Ren,AnnuRevGenomicsHumGenet,2006

SegalE,Nature2006

22

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Kim,T.H.andB.Ren,AnnuRevGenomicsHumGenet,2006

SegalE,Nature2006

23

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer potentialIf active enhancers have promoter activities, it follows thatwhat we classically refer to as promoters could also func-tion as enhancers in certain situations. Although in gener-al a neglected topic of research, we find some clues tosupport this in studies of larger chromatin architectures.

Regulatory elements often interact in close physicalproximity in RNAPII foci that are sometimes referred toas transcription factories [37], and the activity of eachregulatory element within these foci can be influencedby other regulatory elements involved (Figure 1C). Al-though the regulatory mechanisms remain poorly charac-terized, a common model posits that regulatory sequencessynergistically achieve high levels of transcriptional activ-ity by increasing the local concentration of factors neededfor transcription [3741] (Figure 1C). Studies based onchromatin conformation capture have observed RNAPIIfoci involving multiple gene promoters, with few or noenhancers [29,42]. In such arrangements, the degree towhich each gene promoter acts as a promoter or enhancermay depend on the local context of chromatin [42],expressed TFs, and sequence context (e.g., the occurrenceand strength of core promoter elements and TF bindingsites). Using in vitro reporter assays, Li et al. found thatcertain gene promoters in such constellations in MCF7cells had enhancer potential [42], suggesting that they

Box 2. Regulatory elements and associated chromatin

modification states

The N-terminal tails of histone proteins H2A, H2B, H3, and H4 withinnucleosomes can be chemically modified. In particular, amino acidresidues can be methylated or acetylated, which correlates withfunctional properties of the chromatin. For instance, H3K27ac (acet-ylation of lysine 27 in histone H3) is correlated with transcriptionalactivity. The locations of histones marked with a certain histonemodification can be measured genome wide using chromatin im-munoprecipitation (ChIP) coupled with either tiling microarrays(ChIP-chip) or sequencing (ChIP-seq) [38]. Profiling of histone mod-ifications in different cell types has enabled systematic inference ofdifferent types of functional genomic entities [12,53,77,78]. Suchstudies have shown that, typically, the nucleosomes flanking activegene promoters are enriched in H3K27ac, H3K4me3 (trimethylationof lysine 4 in histone H3), and, to some degree, H3K4me1 (mono-methylation of lysine 4 in histone H3), whereas enhancers are en-riched in H3K27ac and H3K4me1, but not H3K4me3, or relatively lowlevels thereof. In addition, several marks have been found to localizeat both enhancers and promoters. Most notably, H3K27ac marksactive promoters and a subset of enhancers that appear to be activelyregulating gene transcription, leading to the notion of classifyingregulatory elements into active and poised classes [15,79]. Poisedregulatory elements sometimes carry bivalent marks associated withactivity and repression at the same time, for instance promotershaving H3K4me3 and H3K27me3 (the latter mark is usually found atinactive gene promoters). Collectively, these studies have arguedthat functional elements can be effectively annotated by their distinctpatterns of histone modifications.

TFs

Regulatory ac!vegene promoters and/or enhancers

Core promoter Core promoterNDR

GTFs

RNAPII RNAPII

(A)

(C)

(B)(i) Silent state

Enhancer

RNAPII

Transcrip!on

Gene promoter

(ii) S!mulus-induced enhancer ac!vity

Enhancer

Enhancement

Gene promoter

(iii) Lagged gene ac!va!on

(i) (ii)

Enhancer

Enhancement

Gene promoter

Low

RNAPIItranscrip!on

Low abundanceof factors

Inac!ve regulatoryelement

Ac!ve regulatory elementPromoter strength and/or transcrip!onal level

High

High abundanceof factors

TRENDS in Genetics

Figure 1. Active regulatory elements are divergently transcribed. (A) Both regulatory active gene promoters and gene-distal enhancers are transcribed. RNA polymerase II(RNAPII) recruitment and transcription initiation are mediated by general transcription factors (GTFs) binding core promoter regions in close proximity to flankingnucleosomes. This is facilitated by transcription factors (TFs), which often bind proximal to core promoters. Transcription often initiates divergently and at the boundary ofthe nucleosome-depleted region (NDR). (B) Gene expression is often preceded by, or changes concurrently with, changes in enhancer transcription. In a silent(nonexpressed) state (i), enhancers and promoters may, or may not, bind RNAPII. Upon stimulus (ii), transcriptional activity at enhancers marks regulatory enhancer activitywith local transcription and increases in RNAPII recruitment at the target gene promoter. (iii) Gene expression may lag behind transcriptional activation at enhancers. (C)Chromatin interactions place regulatory elements in close physical proximity. The individual properties of regulatory elements (chromatin characteristics as well as TF andRNAPII recruitment strengths) as well as context-dependent properties (such as promoter competition, insulation, and core promoter specificity) jointly determine theformation of multiple regulatory interactions (Box 1). Via regulatory cooperation, multiple regulatory elements may increase the local concentration of factors (TFs, GTFs,co-activators, and RNAPII) needed for transcription in RNAPII-enriched foci (i) and thereby achieve in aggregate different levels of transcriptional activity compared withRNAPII foci, including fewer regulatory elements (ii). Nucleosome illustrations in (A) reproduced, with permission, from [38]; (C) modified, with permission, from [38].

Opinion Trends in Genetics August 2015, Vol. 31, No. 8

428

Kim,T.H.andB.Ren,AnnuRevGenomicsHumGenet,2006

AnderssonRetal.,GenomeResearch2009

24

88% of known enhancers that were tested [31]. Theseresults suggest that the promoter activity of an enhanceracross cells is a proxy of its enhancer activity. This con-clusion is also supported by the observation that transcrip-tion at enhancers often changes in a stimulus-dependent

manner [3236] and correlates with changes in the tran-scriptional output of target genes [18,21], often with dy-namics that match or precede gene activation [20,33,35](Figure 1B).

Gene promoters have enhancer pote