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Why should we look at the genome in 3D BMC, Nov 27th 2015 Álvaro Martínez Barrio, PhD [email protected] linkedin.com/in/ambarrio @ambarrio

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  • Whyshouldwelookatthegenomein3D

    !BMC,Nov27th2015lvaroMartnezBarrio,[email protected]/in/[email protected]

  • -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

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  • Whyshouldwelookatthegenomein3D

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

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

  • -log 1

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    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