Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription

Article

Simultaneous mapping of 3D structure and nascent

RNAs argues against nuclear compartments that

preclude transcription

Graphical abstract

Highlights

d

RD-SPRITE enables simultaneous mapping of nascent

transcription and DNA structure

d

Pol II transcription occurs within both A and B compartments

and proximal to nucleoli

d

Nascent pre-mRNAs organize within chromosome territories

and A/B compartments

d

Our findings argue against structural domains that preclude

RNA Pol II transcription

Authors

Isabel N. Goronzy, Sofia A. Quinodoz,

Joanna W. Jachowicz, Noah Ollikainen,

Prashant Bhat, Mitchell Guttman

Correspondence

quinodoz@princeton.edu (S.A.Q.),

mguttman@caltech.edu (M.G.)

In brief

Using RD-SPRITE to simultaneously map

3D genome structure and nascent RNA

transcription genome-wide, Goronzy

et al. find that RNA polymerase II

transcription can occur within multiple

compartments in the nucleus, including

regions previously associated with

inactive transcription, such as the B

compartment and close to the nucleolus.

Goronzy et al., 2022, Cell Reports

41

, 111730

November 29, 2022

ª

2022 The Authors.

https://doi.org/10.1016/j.celrep.2022.111730

ll

Article

Simultaneous mapping of 3D structure and nascent

RNAs argues against nuclear compartments

that preclude transcription

Isabel N. Goronzy,

1,2,3,6

Sofia A. Quinodoz,

1,4,6,

Joanna W. Jachowicz,

1,5,7

Noah Ollikainen,

1,7

Prashant Bhat,

1,2

and Mitchell Guttman

1,8,

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA

David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Present address: Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA

Present address: Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA), 1030 Vienna, Austria

These authors contributed equally

Lead contact

*Correspondence:

quinodoz@princeton.edu

(S.A.Q.),

mguttman@caltech.edu

(M.G.)

https://doi.org/10.1016/j.celrep.2022.111730

SUMMARY

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are

associated with transcriptional activity/inactivity. However, whether these structures are simply correlated

with gene expression or are permissive/impermissible to transcription has remained largely unknown

because we lack methods to measure DNA organization and transcription simultaneously. Recently, we

developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization

of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-

mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments—with

multiple active genes simultaneously colocalizing within the same B compartment—and at genes proximal

to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive

does not preclude transcription and that active transcription can occur throughout the nucleus. In general,

we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure

and transcription.

INTRODUCTION

The three-dimensional (3D) arrangement of DNA in the nucleus is

thought to be important for regulating critical nuclear processes

such as DNA replication and transcription.

1–3

Accordingly, there

have been significant efforts to map DNA structure across

different cell types using proximity ligation methods like 3C,

Hi-C,

5–7

and related variants.

These methods have identified

several structural features including chromosome territories,

A/B compartments, topologically associating domains (TADs),

loops, and enhancer-promoter interactions. However, which of

these are critical for gene regulation and other cellular functions

remains unclear.

The main reason that structure-function relationships within

thenucleus arepoorlyunderstoodisthatcurrentmethodscannot

simultaneously measure transcriptional states and 3D genome

organization.

Instead, analysis of the functional conse-

quences of nuclear structure relies on correlations between

distinct measurements of DNAorganization and gene expression

profiles generated from a combination of experimental methods

(e.g., Hi-C and RNA-seq) in different populations of cells. These

measurements capture an ensemble of many individual cells,

each of which may contain heterogeneous functional states

and structures, making the direct comparison between 3D

structure and transcription challenging.

To highlight this limitation, consider A and B compartments,

which refer to alternating sets of DNA regions that broadly

partition chromosomes; DNA regions within one compartment

preferentially interact with each other (e.g., A-A) rather than

with neighboring regions of the other (e.g., A-B). Early studies

found that A compartments are enriched for genomic DNA re-

gions containing actively transcribed RNA polymerase II (Pol II)

genes, whereas B compartments are depleted for active Pol II

genes and enriched for repressive chromatin marks.

such, these compartments are generally thought to represent

spatial organization of transcriptionally active (A) and inactive

(B) Pol II genes within distinct regions of the nucleus.

13–15

In contrast to this general observation, there are specific genes

located within B compartments that are actively transcribed.

This is predominantly explained by a model where actively tran-

scribed DNA loci ‘‘loop out’’ of the inactive (B) compartment to

localize within an active (A) compartment.

16–19

In this model,

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PolIItranscriptiondoesnotoccurwithinBcompartments;actively

transcribed genes may appear to be within them simply because

of the ensemble nature of compartment (e.g., Hi-C, SPRITE) and

gene expression measurements (e.g., RNA-seq). In support of

this ‘‘looping out’’ model, single-cell microscopy measurements

have shown that individual active genes can be located away

from the remainder of the chromosome from which they are

transcribed,

18–20

that the promoter regions of active genes in

B compartments can form local associations with the A compart-

ment,

and that transcribed genomic loci (measured by interac-

tions between pre-mRNAs) do not form A/B compartments.

Yet, there are other observations to suggest that transcription

may occur within both A and B compartments: many A/B

compartment boundaries remain the same between distinct

cell states despite major changes in gene expression pro-

grams,

and direct recruitment of various gene loci to the nu-

clear lamina (a compartment associated with transcriptional

silencing and located within B compartments) does not always

lead to transcriptional repression for all genes.

22–25

Accordingly,

whether localization of genes within B compartments or other

nuclear structures that have been associated with inactive Pol

II transcription and repressive heterochromatin (such as the

nucleolus and nuclear lamina)

precludes Pol II transcrip-

tion or is simply correlated with inactive transcription remains

unclear.

Recently, we developed RNA & DNA SPRITE (RD-SPRITE),

which enables simultaneous multiway measurements of DNA

and RNA organization in the nucleus.

In our previous study,

we focused on the spatial localization of ncRNAs and their roles

in seeding nuclear organization. However, RD-SPRITE also

measures localization of mRNAs, including individual nascent

pre-mRNAs at their transcriptional loci. Because RNA represents

the functional output of transcription, this approach allows us to

directly measure both 3D genome organization and transcription

at the same location within the nucleus. Here, we show that RD-

SPRITE can be used to assess the relationship between

structural organization and transcriptional activity within different

structural compartments.

RESULTS

RD-SPRITE measures nascent and mature mRNAs at

precise locations in the cell

RD-SPRITEusessplit-and-poolbarcodingtomeasurethespatial

organization of individual RNA and DNA molecules within the cell.

The fundamental measurement unit of RD-SPRITE is the SPRITE

cluster, which contains multiple RNA and DNA molecules that are

in close proximity within a single cell.

Using these clusters,

we can measure multiway RNA and DNA contacts, including

RNA-RNA, RNA-DNA, and DNA-DNA contacts, within higher-or-

der structures in the cell (

Figure 1

A). We previously showed that

RD-SPRITE can accurately measure the 3D spatial organization

of DNA and RNA in the nucleus, including DNA structures such

as chromosome territories, A/B compartments, TADs, and loops

as well as DNA and RNA within nuclear bodies such as the

nucleolus, nuclear speckles, and histone locus body.

Here, we sought to explore whether RD-SPRITE can measure

the 3D organization of distinct populations of mRNAs—including

nascent and mature mRNAs—and their quantitative levels at

various locations in the cell. To do this, we examined the RNA-

RNA and RNA-DNA contacts in our RD-SPRITE dataset

collected from mouse embryonic stem cells. Specifically, we

focused on intronic reads as a surrogate for nascent pre-mRNAs

and exonic reads as a surrogate for mature mRNAs. We

reasoned that newly transcribed (nascent) pre-mRNAs should

be preferentially located on chromatin in proximity to their

genomic DNA locus, while fully spliced (mature) mRNAs should

be associated with ribosomal RNAs (rRNAs) in the cytoplasm.

Consistent with this, we find that intronic reads in RD-SPRITE

represent nascent pre-mRNAs in that they are (1) enriched on

chromatin, (2) enriched for contacts with various small nuclear

RNAs (snRNAs) such as U1 and U2, which are involved in pre-

mRNA splicing in the nucleus, and (3) depleted for contacts

with cytoplasmic RNAs, such as rRNAs (

Figure 1

B). In contrast,

exonic reads show properties consistent with mature mRNAs in

that they are (1) depleted on chromatin, (2) depleted for contacts

with snRNAs, and (3) enriched for contacts with rRNAs (

Fig-

ure 1

B). Together, these data demonstrate that RD-SPRITE

can detect both classes of mRNAs located in different parts of

the cell and distinguish between their localization patterns.

We next tested whether RD-SPRITE can quantitatively mea-

sure the relative abundance of these distinct mRNA populations.

First, we measured whether the overall RNA levels measured by

RD-SPRITE correlate with total RNA-seq measurements and

observed a strong correlation between the levels of RNAs

measured by each approach (Spearman p = 0.79) (

Figure 1

C).

Next, we focused specifically on nascent pre-mRNA levels by

comparing transcription levels estimated from intronic reads in

RD-SPRITE and found them to be highly correlated with those

estimated from global run-on and sequencing (GRO-seq) as-

says,

which measure transcription levels of mRNAs (Spearman

p = 0.86). Finally, we observed a strong correlation between

exonic reads measured by RD-SPRITE and mature mRNA levels

measured by polyA-selected RNA-seq (Spearman p = 0.88).

To confirm the localization of nascent RNAs at their genomic

loci, we measured the DNA contacts of pre-mRNAs (RNA-DNA

contacts) and found them to be enriched for contacts with their

genomic loci (

Figures 1

D and 1E). Next, we explored whether

RD-SPRITE can detect the 3D structure at these actively

transcribing DNA loci. To do this, we mapped the DNA-DNA

contacts of SPRITE clusters containing a specific nascent pre-

mRNA and found that the DNA contacts are highly enriched

surrounding the locus from which the pre-mRNA is transcribed

(

Figure 1

F).

Taken together, these results demonstrate that RD-SPRITE

accurately distinguishes distinct populations of mRNAs within

the cell, enables quantitative measurement of their transcription

levels, and detects the genomic contacts and 3D structure

around individual pre-mRNAs.

Genomic DNA located within B compartments can be

actively transcribed

Because RD-SPRITE accurately measures both nascent RNA

transcripts and higher-order DNA organization genome-wide,

we used it to explore the global structure of genomic DNA

regions undergoing Pol II transcription. Specifically, we

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generated a genome-wide DNA-DNA contact matrix using

SPRITE clusters containing nascent pre-mRNAs. We reasoned

that if most genes within B compartments loop out and

reposition into A compartments when actively transcribed (the

‘‘looping out’’ model), then wewould seea single active compart-

ment in the DNA-DNA contact matrix of actively transcribed re-

gions. Conversely, if genes are transcribed within B compart-

ments, then we would observe both A and B compartments

within this DNA-DNA contact matrix (

Figure 2

A). In fact, the

genomic DNA structures generated from only actively tran-

scribed clusters show clear chromosome territories and intra-

compartment structures comparable to those observed when

measuring DNA-DNA contacts across all SPRITE clusters (

Fig-

ure 2

B). The A/B compartment structure seen in transcribed clus-

ters closely corresponds to A/B compartments defined using

principal eigenvector analysis on the DNA contacts measured

from all SPRITE clusters (

Figure S1

STAR Methods

). This sug-

gests that genes in the B compartment do not ‘‘loop out’’ as

they are transcribed but instead remain in the B compartment.

While it is commonly described as a single compartment, the B

compartment is in fact heterogeneous. Compartment structures

can also be defined using five sub-compartments, three of which

(B1, B2, B3) are considered B-like but differ in repressive

chromatin modifications, gene density, and nuclear location

(

Figure 2

C); B2 and B3 are highly enriched for chromatin features

associated with transcriptional repression, while B1 has

chromatin features more closely resembling the A2 sub-

compartment. Because of this, we considered the possibility

that our observations of transcription within the B compartment

might be restricted to B1. To explore this, we focused on a set of

highly expressed nascent pre-mRNAs in RD-SPRITE and found

these genes to be located within all three B sub-compartments

RD-SPRITE All RNA counts (log

)

RNA-seq counts (log

)

RD-SPRITE Intron counts (log

)

GRO-seq counts (log

)

RD-SPRITE Exon counts (log

)

polyA RNA-seq counts (log

)

-5

Distance from Locus (Mb)

RNA-DNA Contacts (x

)

Intron

Exon

Intron

(nascent)

Exon

(mature)

Intron

(nascent)

Exon

(mature)

Intron

(nascent)

Exon

(mature)

121kb

Gli2

121kb

117kb

Gli2

Nucleus

SPRITE cluster

Gli2

Pola1

Rbfox2

Tet1

Etl4

Rere

ff1

Exoc4

Pias2

Glis

RNA-DNA Contacts

12345

11 12 13 14 15

Cytoplasm

DNA-DNA

pre-mRN

Contacts

Nascent RNA

Mature RNA

Total RNA

mRNA

AAA

rRNAs

Nascent RNA

nascent RNA

DNA

Pol II

snRNAs

SPRITE cluster

Intron

Exon

DNA

snRNAs

DPM

RPM

mRNA

rRNAs

Mature RNA

Figure 1. RD-SPRITE measures nascent and mature mRNAs at precise locations in the cell

(A) Schematic of nascent pre-mRNAs (blue), mature mRNAs (red), and their respective molecular interactions mapped using RNA & DNA SPRITE. Zoom-ins s

how

nascent pre-mRNA contacts in the nucleus (top) and mature mRNA contacts in the cytoplasm (bottom). The specific RNA (RPM) or DNA (DPM) molecules

measured within RD-SPRITE clusters are shown in the dotted circles.

(B) Contact frequency enrichment scores of introns (blue) or exons (red) with chromatin (left), snRNAs (middle), or rRNAs (right) measured using RNA

-DNA or RNA-

RNA interactions.

(left), RD-SPRITE introns and GRO-seq

(middle), and RD-SPRITE exons and polyA-

selected RNA-seq (right).

(D) Aggregated total RNA-DNA contacts of introns or exons with DNA regions surrounding their genomic loci. Shown is the total weighted contact freque

ncy of all

RNAs within these populations contacting 1 megabase (Mb) genomic DNA windows from 10 Mbs up- and downstream of the transcriptional start site.

(E) Examples of weighted RNA-DNA interactions for selected pre-mRNAs (1-Mb resolution). The genomic locus for each pre-mRNA is annotated on the x axi

(F) Weighted DNA-DNA interactions for transcriptionally active loci at the

Gli2

gene locus on chromosome 1 (100-kb resolution). Interactions of transcriptionally

active loci are defined as the DNA contacts occurring within multiway SPRITE clusters containing both nascent Gli2 pre-mRNA transcripts and multiple

DNA

reads.

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Mllt10

Arhgap21

Abi1

Odf2

Sptan1

Tbc1d13

Gtdc1

Mbd5

Rif1

chr 2

10Mb

53Mb

RNA-DNA contacts

Normalized contacts

DNA Compartment on Chr2

1.3

1.2

1.1

1.0

0.9

A / B pre-mRNA-DNA

inter-comp. domain enrichment

0 25 50 75 100 125 150 175

Position on chr 2

-0.75

-0.5

-0.25

0.0

0.25

0.5

0.75

1.0

1.25

A / B pre-mRNA-DNA inter-comp.

domain enrichment (rank mapped)

Weighted contacts

chromosome 2

200

175

100125

150

chr 2

Weighted contacts

100

350

200 250

150

300

chr 2

Weighted contacts

300

150 200

100

250

chromosome 2

chr 2

B2/B3 Transcribed clusters

A2/B1 Transcribed clusters

A1 Transcribed clusters

A compartment B compartment

chromosome 4

Weighted contacts

0 100 200 300 400 500

Weighted contacts

0 400 800 1200

DNA-DNA contacts

Chr 2

Weighted contacts

450

0350

150 250

0400

Weighted contacts

1200

800

A compartment B compartment

DNA-DNA contacts

Chr 4

Expected

DNA-DNA

Heamaps

Sub-Compartment

DNA-DNA contacts: per subcompartment

DNA-DNA

pre-mRNA

DNA-DNA

pre-mRNA

DNA-DNA

pre-mRNA

Mean

snRNA

% Nucleolar

Hub Regions

% Speckle

Hub Regions

% Genome

% Matching

A/B Labels

Mean Gene

Density

Mean

pre-mRNAs

Numb. Top

2000 genes

A1 A2

B1 B2

0.15 0.13 0.16

0.29 0.27

0.94

0.41

0.67 0.96 0.95

2.99 1.76 1.49

0.65 0.67

353 230

204 103 94

100

53 47 23 21

870 467 467

97 58

0.91

0.03 0.06 0 0

0.44

0.18 0.12

0.04

0.23

Min

Max

Mllt10

Arhgap21

Abi1

Odf2

Sptan1

Tbc1d13

Gtdc1

if1

A compartment B compartment

All DNA

Transcribed DNA

All DNA

Transcribed DNA

“Looping Out” Model:

Transcription only in

A Compartment

“Permissive” Model:

Transcription in both

A and B Compartments

Off

x10

-3

All

Figure 2. Genomic DNA located within B compartments can be actively transcribed

(A) Two models of RNA Pol II gene transcription within A or B compartments and the expected DNA-DNA interaction matrices for actively transcribed loci

. The

‘‘looping out’’ model requires B compartment genes to loop into the A compartment to be transcribed, and the corresponding DNA-DNA matrix generated f

rom

transcribed DNA regions (left heatmap, upper diagonal) would not have compartment structure, while the heatmap generated from all DNA regions would

have

compartment structure (lower diagonal). In the ‘‘permissive’’ model, transcription of B compartment genes occurs without a change in genomic struc

ture, and the

corresponding DNA-DNA matrix from transcribed DNA regions (right heatmap, upper diagonal) would have A/B compartment structure.

(B) Weighted DNA-DNA interaction heatmaps for SPRITE clusters containing nascent pre-mRNAs (upper diagonal) versus all SPRITE clusters (lower dia

gonal).

Chromosomes 2 and 4 are shown as examples.

(C) Feature profiles of A/B sub-compartments at 100-kilobase (kb) resolution. Characteristics include percentage (%) of genome assigned to each sub

compartment (top), % of 100-kb regions for each sub-compartment matching the corresponding ‘‘super-compartment’’ labels (i.e., A1-A, B1-B) calcu

lated by

principal eigenvector analysis of RD-SPRITE (second), mean number of protein-coding genes per 100-kb DNA region (third), mean weighted RNA-DNA con

tacts

of pre-mRNAs per 100-kb DNA region (fourth), mean weighted RNA-DNA contacts of small nuclear RNAs (snRNAs) per 100-kb DNA region (fifth), number of top

2,000 genes within each sub-compartment (sixth), and percentage of speckle or nucleolar hub regions within each sub-compartment (seventh and eight

h).

(D) Weighted DNA-DNA interaction heatmaps for actively transcribing SPRITE clusters containing nascent pre-mRNAs of genes in various sub-com-

partments.

(E) Unweighted RNA-DNA interactions of nascent pre-mRNAs at the B-A-B compartment boundaries near the front end of chromosome 2 (10–53 Mb).

(legend continued on next page)

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(

Figure 2

STAR Methods

). Focusing specifically on the sub-

compartments associated with repressive features (B2 or B3),

we measured the DNA organization (DNA-DNA contacts) when

pre-mRNAs are actively transcribed. We selected individual

SPRITE clusters that contain reads for nascent pre-mRNAs

located within B2 or B3 and generated a DNA-DNA heatmap

(

Figure 2

D). We found that actively transcribed genomic

regions within these sub-compartments maintain DNA-DNA

contacts with other B2 and B3 regions and do not contact neigh-

boring A1 sub-compartment genomic regions. Conversely,

when we used clusters containing pre-mRNAs from genes within

the A1 sub-compartment to generate a DNA-DNA heatmap, we

observed preferential contacts with other A1 regions but not

contacts with neighboring B compartment DNA regions.

Together, these results demonstrate that active transcription

can occur within all B sub-compartments.

TofurthervalidatethatBcompartmentstructuresareobserved

when B compartment genes are actively transcribed, we

explored the DNA contacts of nascent pre-mRNAs. RD-SPRITE

detects long-distance RNA-DNA interactions between nascent

pre-mRNAs and genomic DNA sites beyond their transcriptional

loci(

Figure1

E).ToinvestigatetheunderlyinggenomicDNAstruc-

ture during active transcription of B compartment genes, we

looked for A/B compartment structures in these long-range

RNA-DNA contacts. Indeed, beyond RNA-DNA contacts

between pre-mRNAs and their own loci, B compartment pre-

mRNAs are enriched for contacts with DNA regions located in

neighboring B compartments and depleted for contacts with

DNA regions located within A compartments (

Figures 2

E–2G).

Because these nascent RNA transcripts are located near their

gene locus, this confirms that genes contained within B

compartments do not ‘‘loop out’’ when transcribed.

Together, these results indicate that localization of genes

within B compartments does not preclude transcription.

Nascent pre-mRNAs organize within genome-wide

structures resembling A/B compartments

We next wondered whether multiple, simultaneously transcribed

genes organize together within the B compartment. To explore

this, we generated an RNA-RNA contact matrix to measure the

genome-wide spatial organization of nascent pre-mRNAs

(

Figures 3

A and 3B). Because the number of observed RNA con-

tacts is dependent on expression level, we focused on the 2,000

most highly expressed genes to ensure high-confidence mea-

surements of individual pre-mRNA contacts (

Table S1

). These

highly expressed genes include those located within both A

and B compartments (1,216 A genes and 784 B genes) and

display comparable expression levels (

Figures S1

C and

A).

We sorted these pre-mRNAs by the genomic position of their

gene locus and observed clear structural patterns, including

the following: (1) preferential contacts between pre-mRNAs

that are transcribed from the same chromosome, reminiscent

of chromosome territories (

Figure 3

A), and (2) alternating blocks

of highly interacting pre-mRNAs within individual chromosomes,

reminiscent of A/B compartments (

Figure 3

B). In contrast, con-

tact matrices generated between mature mRNAs (exons) do

not display preferential contact frequencies based on their

genomic positions, consistent with their localization in the

cytoplasm (

Figures S3

A and S3B).

To determine whether these intrachromosomal structural

patterns correspond to A/B compartments, we compared

them to the 3D structure of their corresponding genomic DNA

loci. We generated a DNA-DNA contact matrix for these highly

expressed genes (gene-level heatmap) and observed highly

similar intrachromosomal patterns in the DNA-DNA and the

pre-mRNA RNA-RNA contact maps (Pearson r = 0.83), but

not between gene-level DNA and mature mRNA contact maps

(Pearson r = 0.04) (

Figures 3

Cand

C). Next, we defined A/B

compartments using nascent RNA-RNA contacts and asked

whether their quantitative (eigenvector) values matched those

defined using DNA-DNA contacts. First, we ensured that A/B

compartment scores based on the gene-level DNA-DNA con-

tacts were similar to those measured across the genome (Pear-

son r = 0.87,

STAR Methods

) to confirm that this gene-level

analysis is comparable to genome-wide analysis (

Figure 3

D).

Second, we compared the gene-level DNA-DNA eigenvectors

to those calculated from the nascent RNA-RNA contact matrix

and found a strong correlation (Pearson r = 0.75) (

Figure 3

E).

Finally, we grouped RNA-RNA contacts based on A/B compart-

ment definitions from genomic DNA and found that pre-mRNAs

transcribed from B compartments display a high contact

frequency with other pre-mRNAs transcribed from B compart-

ments, but not with pre-mRNAs transcribed from loci contained

within A compartments, and vice versa (

Figure 3

F). In contrast,

mature mRNAs do not display any preferential interactions

between A/B regions (

Figures S3

D–S3F).

To ensure that the observed compartmentalization of nascent

RNAs is not a unique feature of highly expressed genes, we

explored compartmentalization properties across mRNAs that

span a broad range of expression levels (i.e., the top 10,000

most abundant pre-mRNAs) (

Figure S4

A) and observed prefer-

ential A-A and B-B contacts and depletion of neighboring A-B

contacts (

Figure S4

B). Indeed, zooming in on chromosome 2,

we detected clear B-A-B compartment structures, comparable

to those measured for the most abundant 2,000 pre-mRNAs,

within the RNA-RNA contacts of lower expression genes (

Fig-

ure S4

C). This indicates that the organization of pre-mRNAs

within A/B compartments and transcription within the B

compartment is observed across a range of expression levels

and all classes of transcribed Pol II genes.

These results are consistent with our observations that

actively transcribed genes are spatially organized into A/B

compartments and that multiple genes are simultaneously tran-

scribed within B compartments (

Figure 3

G). If transcription only

(F) Inter-compartment pre-mRNA-DNA contact enrichment score for A versus B compartment genes (black solid line) and the first eigenvector (E1) (gray

dotted

line) along chromosome 2. Enrichment scores were rank-remapped to E1 for direct comparison (

STAR Methods

(G) Mean inter-compartment pre-mRNA-DNA enrichment scores for A versus B compartment genes on A (left) or B (right) compartment genomic regions of

chromosome 2. Error bars show 95% bootstrapped confidence intervals.

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