Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 4, pp. 688-700 © Pleiades Publishing, Ltd., 2024.

688

REVIEW

Nonspecific Interactions in Transcription Regulation

and Organization of Transcriptional Condensates

Anna A. Valyaeva

1,2,3,a

* and Eugene V. Sheval

2,3

Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119991 Moscow, Russia

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Department of Cell Biology and Histology, Faculty of Biology, Lomonosov Moscow State University,

119991Moscow, Russia

e-mail: valyaeva.ann@gmail.com

Received October 10, 2023

Revised November 19, 2023

Accepted November 20, 2023

Abstract— Eukaryotic cells are characterized by a high degree of compartmentalization of their internal contents,

which ensures precise and controlled regulation of intracellular processes. During many processes, including

different stages of transcription, dynamic membraneless compartments termed biomolecular condensates are

formed. Transcription condensates contain various transcription factors and RNA polymerase and are formed by

high- and low-specificity interactions between the proteins, DNA, and nearby RNA. This review discusses recent

data demonstrating important role of nonspecific multivalent protein–protein and RNA–protein interactions in

organization and regulation of transcription.

DOI: 10.1134/S0006297924040084

Keywords: transcription, RNA, RNA polymerase, transcriptional condensates, intrinsically disordered regions

Abbreviations: ARM, arginine-rich motif; CTD,C-terminal do-

main; DFC,dense fibrillar component; GC,granular compo-

nent; IDR, intrinsically disordered region; mRNA, messen-

ger RNA; PRC2, Polycomb repressive complex 2; RNAPI,RNA

polymerase I; RNAPII, RNA polymerase II; RNAPIII, RNA

polymerase III; rRNA,ribosomal RNA; tRNA, transfer RNA.

* To whom correspondence should be addressed.

INTRODUCTION

Gene expression in the nucleus is orchestrated

by the coordinated activity of three RNA polymerases:

RNA polymerase I (RNAPI), which is responsible for

transcribing the precursor of 18S, 5.8S, and 25S ribo-

somal RNA(rRNA); RNA polymerase II(RNAPII), which

is predominantly involved in transcribing messenger

RNAs(mRNAs); and RNA polymerase III(RNAPIII), which

is responsible for transcribing ribosomal 5S rRNA, trans-

fer RNA (tRNA), 7SL RNA, and various other small RNAs.

Regulation of transcription is a complex process that

relies on the intricate interplay of numerous proteins

and RNAs that accumulate at specific loci in the nu-

cleus. An illustrative example of such accumulation

is nucleolus, the largest organelle within the nucleus,

where the processes of transcription, pre-mRNA pro-

cessing, and pre-ribosomal assembly take place[1, 2].

The key feature of the cell nucleus, which en-

ables transcription and its flexible regulation, is high

mobility of nuclear proteins. In 2000, data began to

accumulate indicating that many proteins in the nu-

cleolus, interchromatin granules, and chromatin are

not stably bound to the structures, but rather are in

continuous and relatively rapid exchange with the sur-

rounding nucleoplasm [3, 4]. Focus of this review is on

the nature of binding and exchange of the transcrip-

tion factors responsible for carrying out the relatively

long process of transcription. While constant binding

of transcription factors to promoters could theoreti-

cally activate transcription, early experiments showed

that the glucocorticoid receptor, a hormone-dependent

modulator of gene expression, undergoes rapid ex-

change between the chromatin target site and the sur-

rounding nucleoplasm [3]. It was later shown that both

the glucocorticoid receptor and its interacting partner,

glucocorticoid receptor interacting protein 1 (GRIP-1),

exist in a dynamic equilibrium with the promoter and

must repeatedly return to the DNA template during

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

transcription activation [5]. Other transcription factors

also exhibit dynamic interactions with promoters, oc-

cupying the promoter region for relatively short peri-

ods of time ranging from fractions of a second to tens

of seconds [6]. RNAPII subunits have also been shown

to dynamically assemble at the promoter site [7].

High exchange rates have also been demonstrat-

ed for some rRNA transcription factors – UBF [8-10],

TAFI48, PAF53, and TIF-IA/Rrn3 [9]. RNAPI subunits

also exhibited dynamic behavior at the promoter, sug-

gesting stochasticity and low efficiency in the assem-

bly of complexes required for transcription [9].

Behavior of transcription factors is described by

the 3D genome-scanning model [6], also referred to as

the diffusion and affinity model. This model assumes

that proteins diffuse freely and rapidly throughout

the nucleus, intermittently interacting with its compo-

nents. Most of these interactions are nonspecific and

non-functional. However, when proteins engage with

the target sites through high-affinity interactions, they

could become temporarily immobilized. This, with a

seemingly low probability, may result in formation of

the functional macromolecular complexes.

However, this model fails to describe all quantita-

tive regularities associated with regulation of the ge-

nome activity. For example, it has been calculated that

the rate at which proteins locate their sites on DNA in

bacterial cells is approximately 100 times higher than

predicted based on diffusion alone [11, 12]. Several

mechanisms have now been proposed that explain

enhanced efficiency and accuracy of protein accumu-

lation. At least some of these mechanisms rely pre-

dominantly on low-specificity interactions between

molecules, leading, in particular, to formation of the

membraneless structures now commonly referred to

as biomolecular condensates. These condensates serve

as repositories for the molecules essential for efficient

transcription. Given that formation of condensates in

many cases depends on the interaction between pro-

teins and RNAs, this review will systematically explore

the roles of proteins and RNAs involved in organiza-

tion and regulation of transcription through formation

of transcriptional condensates.

COMPARTMENTALIZATION

OF TRANSCRIPTION

Regulation of gene transcription is primarily

based on numerous high-affinity (specific) interactions

of various proteins with enhancers and promoters. For

example, transcription activation depends on the in-

teraction of the Mediator complex with the enhancer

and promoter, which facilitates landing of RNAPII

during transcription initiation [13, 14]. However, it has

been suggested in a number of studies that transcrip-

tion initiation can lead to formation of rather large

complexes – transcription factories – in which many

transcribed genes are concentrated. This hypothesis is

compelling as it holds the promise of coordinated reg-

ulation for numerous genes within a single transcrip-

tion factory. Existence of such complexes is primarily

supported by the microscopic data showcasing clus-

tering of RNAPII molecules in animal nuclei [15-20].

These observations were initially made using conven-

tional fluorescence microscopy rather than super-res-

olution localization microscopy, which allows precise

determination of the number of molecules in a given

area. The results obtained using the super-resolution

microscopy indicate that transcription factories are

relatively small and are predominantly composed of

only a few RNAPII molecules [21].

Subunits of the Mediator complex are also found

among the constituents of the transcription factories

[22]. It has been suggested that a fraction as small as

<10% of all clusters consisting of large assemblies of

RNAPII and Mediator may correspond to the previous-

ly described clusters of super-enhancers. Regulation

of certain crucial genes involves multiple enhancers,

known as super-enhancers [23-27]. Existing data indi-

cate that the large clusters of RNAPII and the Mediator

complex often are co-localized with the loci containing

super-enhancers [22, 28-30].

Live cell observations suggest that such transcrip-

tion factories are formed as dynamic, short-lived aggre-

gates that include RNAPII molecules [31-33]. A similar

situation has been demonstrated for the transcription

factors Zelda and Bicoid in the developing Drosophila

embryos [34]. Both of these factors generate dynamic,

short-lived condensates that transiently interact with

active sites of Bicoid-dependent transcription thus ac-

tivating it.

Finally, microscopic visualization of the transcrip-

tion of long genes should be considered. Long genes

form rather extended loops along which RNAPII mol-

ecules appear to move [35, 36]. These loops share sim-

ilarities with the structures observed in lampbrush

chromosomes [37-39]. Consequently, recent data do

not align with the concept of highly ordered transcrip-

tion factories; instead, they suggest the existence of

highly dynamic complexes. These dynamic complexes

are now commonly referred to as transcriptional con-

densates [40].

ROLE OF NONSPECIFIC INTERACTIONS

IN THE FORMATION

OF TRANSCRIPTIONAL CONDENSATES

The concept of biomolecular condensates originat-

ed in the study of membraneless organelles [41, 42].

Subsequently, this concept was extended to any com-

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 1. Role of intrinsically disordered domains (IDRs) in formation of biomolecular condensates. a)Electrostatic interactions

between the protein IDRs in the presence of domains with different charges. b)Interactions between the protein IDRs and RNA.

c)Formation of a biomolecular condensate due to multivalent interactions provided by the dimers of proteins with IDRs binding

RNA molecules.

plex that does not exhibit stoichiometry in its composi-

tion [43]. Formation of such complexes, irrespective of

their size, relies on numerous low-affinity interactions

among the molecules. Crucially, interactions involving

intrinsically disordered protein domains (IDRs) play

a pivotal (though not exclusive) role [44-47] (Fig. 1,

a and b). Additionally, multivalent interactions involv-

ing structured domains are of significance [48, 49].

Interms of their role in the condensate formation, pro-

tein molecules are categorized into scaffold molecules,

participating in multivalent interactions and contrib-

uting to condensate formation, and client proteins,

which accumulate within already formed condensates

[49]. Although IDRs do not exhibit high affinity, they,

as well as the high-affinity RNA-binding domains, can

serve as foundation for multivalent interactions –

either due to extensive IDR length or through oligom-

erization of the protein molecules (Fig.1c).

Ability of some proteins involved in transcription

to form condensates in vitro suggests that transcription

may be accompanied by the formation of biomolecular

condensates. The most detailed data are available for

some nuclear proteins, in particular for two pivotal

nucleolar proteins, FBL and NPM1 [50]. Numerous other

proteins engaged in the diverse aspects of genome ac-

tivity regulation demonstrate capability to form con-

densates in vitro. In some instances, the analysis was

limited to the IDR of these proteins, but even such data

appear to be sufficient to indicate potential involve-

ment of the proteins in the biomolecular condensate

formation. This group of proteins include components

of the Mediator complex and RNAPII [22, 28, 29], TAZ

[51], BRD4 [28], and transcription factors OCT4 [52]

and TAF15 [53].

One of the most striking pieces of evidence sup-

porting the formation of the IDR-dependent biomolec-

ular condensates during transcription activation comes

from the study of the Wnt/β-catenin signaling pathway

[54]. Upon pathway activation, β-catenin translocates

to the nucleus, where it forms a complex with the LEF/

TCF transcription factors. This complex recruits addi-

tional cofactors and enhances expression of the target

genes. LEF1 demonstrates the ability to form biomo-

lecular condensates with β-catenin both in vitro and

in vivo, and the formation of these condensates is es-

sential for transcription activation. Notably, LEF1 with

disrupted IDR loses its activity, which can be restored

by substituting with the intrinsically disordered do-

main of another protein (FUS). This emphasized the

significance of nonspecific interactions in the tran-

scription regulation.

Moreover, recent data suggest that IDRs can not

only enhance transcription efficiency by attracting

diverse proteins into the transcriptional condensates,

but also improve accuracy of the promoter recogni-

tion by transcription factors. Transcription factors

possess DNA-binding domains that bind to the specific

short DNA sequence motifs. However, these DNA-bind-

ing domains constitute only a fraction of transcrip-

tion factor sequences, with the majority being of low

complexity and lacking a stable 3D structure [55-57].

As mentioned earlier, IDRs are not capable of high-af-

finity and high-specificity interactions. Nevertheless,

these regions can “target” transcription factors to the

specific genomic loci, thereby increasing specificity of

the promoter recognition, when acting in concert with

high-affinity DNA-binding domains [58-60].

ROLE OF RNAPII C-TERMINAL DOMAIN

IN THE ORGANIZATION

OF TRANSCRIPTIONAL CONDENSATES

C-terminal domain of RNAPII (CTD) plays a dis-

tinctive role in organization of transcription processes.

The CTD consists of numerous heptad repeats with the

consensus sequence Tyr 1-Ser 2-Pro 3-Thr 4-Ser 5-Pro 6-

Ser 7 [61-63], and is characterized as an intrinsically

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

disordered region. Its length exhibits considerable

variation across different organisms, ranging from 26

repeats in Saccharomyces cerevisiae to 52 repeats in

humans.

When expressed in cells, the CTD accumulates at

active transcription sites, indicating its potential role

in the recruitment of RNAPII at target sites [64]. How-

ever, the data regarding its direct involvement in the

transcription process are conflicting. Catalytic activi-

ty of RNAPII relies on the conserved enzyme subunits

RPB1 and RPB2, rather than the CTD [65], so it seems

logical that the RNAPII with deleted CTD can perform

transcription in vitro but not in vivo [66]. Multiple in-

vestigations have explored this issue, with the most re-

cent study utilizing Raji cells suggesting that the CTD

is not required for transcription in live cells [67].

The findings presented in this study suggest that the

CTD could be required not for the transcription itself,

but rather for the precise localization of RNAPII at the

target sites, as well as for regulation of the transcrip-

tion-related processes and transcript maturation.

Both human and yeast CTD sequences are able to

form condensates in vitro, resulting in spherical aggre-

gates that contain intact RNAPII [68]. Importantly, the

ability of RNAPII to cluster in the nucleus correlates

with the number of heptad repeats: shortening the

human CTD to the length of the yeast CTD reduces

concentration of the accumulated RNAPII and its as-

sociation with chromatin in human cells. Conversely,

increasing the CTD length enhances clustering of the

RNAPII molecules [69]. Modeling confirms that the CTD

length promotes binding of RNAPII molecules to the

promoter and delays their release from it, while the

CTD–CTD interactions facilitate concentration of nu-

merous RNAPII molecules at the single point. Several

studies also suggest that CTD is involved in the recruit-

ment of the Mediator complex [22, 28, 70]. Disruption

of the Mediator complex results in disassembly of the

large complexes of hypophosphorylated RNAPII [71].

The studies cited above suggest that formation of

biomolecular condensates accumulating RNAPII, vari-

ous transcription factors, and other proteins could rep-

resent a crucial mechanism for enhancing transcrip-

tion processes. However, this mechanism might be less

effective in S. cerevisiae. The CTD in this organism is

much shorter than, for example, in humans, where

the longer CTD leads to the stronger CTD–CTD interac-

tions and formation of the less dynamic condensates

[68]. This distinction could potentially explain why the

RNAPII molecules in S. cerevisiae nuclei do not form

clusters, a characteristic feature of the human cell nu-

clei [72]. Observations of individual RNAPII molecules

in the living yeast cells demonstrated that CTD plays a

role in restricting diffusion of RNAPII within the nu-

clear region enriched with active genes, although no

visible condensate formation was evident. Shortening

of the CTD resulted in the reduced diffusion restric-

tion, increased target site search time, and impaired

formation of the preinitiation complex. According to

the authors, these differences could be attributed to

the unique organization of transcription within a small

yeast cell nucleus, where all components are concen-

trated in a limited space, making formation of the

transcriptional condensates redundant [72].

Significant insights have been generated using

an optogenetic system designed to induce biomolecu-

lar condensates based on the IDRs of FET family tran-

scriptional regulators [such as Fused in sarcoma (FUS),

Ewings sarcoma (EWS), and TAF15] [73]. These IDRs

can initiate phase separation process in the living cells

with TAF15 demonstrating capacity to interact with

the RNAPII CTDs and attract RNAPII molecules to form

condensates. Notably, nascent CTD clusters at primed

genomic loci lower the energy barrier for growth of

the TAF15 condensates, which, in turn, further recruit

RNAPII to initiate transcription. Thus, the authors have

identified a positive feedback mechanism that enhanc-

es RNAPII accumulation at transcription sites, leading

to improved transcription efficiency. At the same time,

the data suggest that elongation occurs outside of the

induced condensates, as the RNAPII molecules with

unmodified CTD colocalize with the condensates con-

sisting of IDRs of TAF15, and phosphorylated CTD is re-

leased into the surrounding region.

Therefore, the existing data demonstrate that non-

specific interactions involving the disordered CTD of

RNAPII can enhance transcription efficiency. It is cru-

cial to note, however, that many of the data providing

information on the mechanisms underlying formation

of transcriptional condensates and role of IDRs are

derived from the in vitro experiments or experiments

with isolated IDRs, rather than the whole proteins.

Within the intact proteins, IDRs may exhibit different

behavior. For example, recent findings suggest that the

IDRs alone could be insufficient for inducing transcrip-

tion factor clustering [74], so this issue needs further

investigation.

TWO TYPES OF TRANSCRIPTIONAL

CONDENSATES PROVIDE SEPARATION

OF INITIATION AND ELONGATION PROCESSES

Recent advancements in super-resolution micros-

copy techniques enable estimation of the distances

between transcription factors and nascent transcripts.

For example, in the case of the Pou5f1 and Nanog genes,

nascent transcripts were found to be in close proximi-

ty to the elongating RNAPII molecules but distant from

the loci enriched with the SOX2 and BRD4 transcrip-

tion factors [75]. These findings imply spatial separa-

tion between the elongation and initiation complexes.

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Fig. 2. Models of initiation and elongation condensates that provide regulation of transcription by RNA polymeraseII (RNAPII).

Intriguingly, results obtained with the HIV-1 reporter

gene indicate that the initiation complex (initiation

condensate) exists for a relatively short time (~1min),

during which RNAPII molecules are recruited to the

promoter region [76]. Apparently, during transcription

and subsequent processes such as splicing and nuclear

transport, the molecules travel through several con-

densates, ensuring high efficiency at each stage of the

RNA molecule life cycle[77].

To date, sufficiently compelling data confirming ex-

istence of condensates where the components involved

in transcription initiation are concentrated have been

obtained (Fig. 2). The situation regarding existence of

the elongation condensates is somewhat more contro-

versial. As mentioned earlier, long genes form extend-

ed loops along which RNAPII molecules move during

transcription [35, 36]. It is likely that the described

patterns could be extended to all other genes, but this

requires experimental confirmation, focusing on the

key aspects. Nevertheless, these morphological obser-

vations do not align well with the idea of isolated elon-

gation condensates. Further experiments will be able

to provide clarity on these findings and reconcile the

existing contradictions.

ROLE OF NONSPECIFIC INTERACTIONS

IN THE ORGANIZATION

OF THE PRE-rRNA TRANSCRIPTION

Nucleolus is the product of transcription from the

relatively small regions of the genome containing nu-

merous rRNA genes, referred to as nucleolus organizer

regions [78]. Around 80 ribosomal proteins and over

200 pre-ribosome assembly factors accumulate in the

nucleoli [79]. Additionally, nucleolus harbors a sub-

stantial number of other proteins unrelated or only in-

directly related to its primary function– production of

pre-ribosomes [80].

Unlike most of the nuclear bodies, nucleoli exhib-

it relatively complex internal organization. Active nu-

cleoli in mammals, birds, and some reptiles consist of

three distinct subcompartments: fibrillar center (FC),

dense fibrillar component (DFC), and granular com-

ponent(GC), clearly visible in electron microscopy im-

ages [81, 82]. The DFC and GC form during ribosome

biogenesis.

Transcription of rDNA occurs at the boundary of

FC and DFC; consequently, DFC represents a cluster of

proteins involved in the early pre-rRNA processing.

Central role in maintenance of structural integrity of

this nucleolar subcompartment is played by fibrillarin

(FBL), which binds to the pre-rRNA molecules during

transcription [83]. Thus, the transcribed pre-rRNA mol-

ecules nucleate assembly of DFC, where both tran-

scription and initial steps of pre-rRNA processing take

place simultaneously. It must be noted that this pro-

cess relies on the disordered glycine and arginine-rich

N-terminal region of FBL, known as GAR domain [83].

FBL, a highly conserved methyltransferase, possesses an

amino acid sequence and 3D structure that have under-

gone minimal changes from archaea to higher eukary-

otes [84]. Emergence of the GAR domain has enabled

FBL to acquire additional functions essential for its role

in the significantly more complex eukaryotic cells. Spe-

cifically, this domain serves as a nuclear localization

signal [85]. Another potential function is involvement

of the GAR domain in organization of the DFC [83].

GC is traditionally viewed as a compartment dedi-

cated to pre-rRNA processing and pre-ribosome assem-

bly. Structural integrity of the GC relies on dynamic

interactions between the pre-ribosomes with NPM1

playing a pivotal role in this process [86]. During mat-

uration, properties of the GC change to facilitate di-

rected transport of pre-ribosomes from the DFC to the

nucleolus periphery [87].

Similar to the RNAPII transcriptional condensates,

multiple condensates are formed inside the nucleolus,

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

determining directional movement of the synthesized

pre-rRNA molecules.

ROLE OF THE SYNTHESIZED TRANSCRIPTS

IN THE REGULATION OF GENE EXPRESSION

Initiation of phase separation processes during

the biomolecular condensate formation often relies on

the RNA molecules serving as successful substrates for

multivalent interactions. The subsequent discussion

will highlight key studies suggesting involvement of

RNA molecules in the low-specificity interactions cru-

cial for transcription regulation.

Variety of RNAs and transcription process itself

influence three-dimensional structure of chromatin

and gene expression mediated by the chromatin state

[88-92]. Active transcription and accumulation of the

chromatin-associated transcripts at the gene locus, in-

cluding pre-mRNAs and other nascent RNAs, maintain

open state of the chromatin through a positive feed-

back mechanism. Transcripts regulate chromatin state

and associated gene expression through electrostatic

interactions with the similarly charged DNA and oppo-

sitely charged histone proteins [93]. Additionally, they

bind to various protein factors comprising chroma-

tin[94].

Transcription and chromatin remodeling factors

YY1, CTCF, DNMT1, and DNMT3A exemplify DNA-bind-

ing factors whose association with chromatin can be

regulated by nascent transcripts [95]. For the ubiqui-

tously expressed transcription factor YY1, interaction

with the nascent RNAs stabilizes its binding with chro-

matin, predominantly at promoter sites [96]. In con-

trast, nascent RNAs have an inhibitory effect on asso-

ciation of the DNMT1 and DNMT3A methyltransferases

with chromatin [97, 98]. For the DNMT1, which per-

forms maintenance methylation of CpG islands, inter-

action with various nascent transcripts [99], including

of its own mRNA [100], has been demonstrated using

the whole-genome RNA–protein interactome analysis

methods (fRIP-seq). Interestingly, DNMT1 prefers bind-

ing to the non-canonical G-quadruplex structure called

pUG-fold, with binding affinity appearing proportional

to the length of the GU repeat [100]. Interaction with

the RNA pUG-fold prevents DNMT1 from binding to

DNA, thereby inhibiting its enzyme activity. Converse-

ly, certain long non-coding RNAs attract methyltrans-

ferase to the specific chromatin regions without inhib-

iting its activity [101-105], and could even determine

its cellular localization [106].

Antagonistic relationships with the nascent tran-

scripts are also observed for the Polycomb repressive

complex 2 (PRC2) [107]. PRC2, an epigenetic transcrip-

tion regulation complex, methylates lysine 27 of the

histone H3, participating in the repression of gene

expression. Due to its function, PRC2 is primarily as-

sociated with CpG islands in the repressed genes.

High-throughput sequencing of the RNAs interacting

with PRC2 components (CLIP/iCLIP-seq) revealed PRC2

interactions with the pre-mRNAs of the majority of

active genes [107, 108]. Inhibition of RNAPII or total

RNA degradation in the cell led to the recruitment of

PRC2 to active genes [109-111], with this effect being

reversible [112, 113]. In vitro experiments demonstrat-

ed RNA competition with nucleosomes for PRC2 bind-

ing and inhibition of PRC2 catalytic activity [107, 

114-116]. Interestingly, higher affinity was shown for

the RNAs forming G-quadruplex structures [112, 117].

Bytitrating PRC2 complexes with themselves, nascent

transcripts reduce likelihood of these repressive com-

plexes binding to chromatin, thereby preventing gene

silencing. It has been hypothesized that all PRC2–chro-

matin interactions are mediated by the RNA “bridge”

[118]. However, evidence from the rChIP-seq exper-

iments indicating necessity of RNA presence in the

complex with PRC2 for chromatin localization seems

to be an artifact of the experiment and normalization.

Later studies demonstrated that treating the immuno-

precipitated chromatin with RNase A and sonication

in a low ionic strength solution reduced specificity of

immunoprecipitation and increased background sig-

nal of nonspecific chromatin precipitation [119, 120].

Therefore, active transcription leading to formation of

new transcripts that bind and immobilize repressive

epigenetic regulators maintains itself.

Involvement of the nascent transcripts in regu-

lation of the binding of chromatin-associated protein

factors to chromatin seems to be a more widespread

mechanism for controlling the structure and state of

chromatin than previously thought. For example, ex-

periments involving inhibition of transcription and

RNA degradation in the cells have demonstrated that

disappearance of RNA from chromatin coincides with

the changes in the chromatin-associated proteome

[121, 122]. Transcriptional inhibition and concurrent

decrease in the number of nascent transcripts result

in the recruitment of chromatin modifier proteins and

chromatin remodeling factors (DNA methyltransferas-

es of the DNMT family, EHMT1/2, MLL2/SET1A, HUSH,

NuRD, NURF, NoRC, CHRAC, NuA4, INO80, BAF, ATRX/

DAXX, cohesin, CTCF, SMCHD1, SAFB) and transcrip-

tion factors (POU5F1, ZFP57, UBTF, TP53, MYBL2, and

UTF1) to chromatin. Meanwhile the number of chro-

matin-associated RNA processing factors were lost

from the chromatin during RNAPII inhibition. A sim-

ilar qualitative change in the chromatin-associated

proteome occurs in the case of RNA degradation [121].

Furthermore, significant proportion of the chro-

matin- associated proteins are known as RNA-bind-

ing proteins [122]. These proteins, often have IDRs or

low complexity sequences, and can, through binding

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

to nascent transcripts in close proximity to RNAPII,

facilitate formation of biomolecular condensates via

the phase separation mechanism, promoting concen-

tration of the factors necessary for gene expression at

the transcription site.

ROLE OF THE SYNTHESIZED TRANSCRIPTS

IN THE FORMATION

OF TRANSCRIPTIONAL CONDENSATES

Numerous transcription factors interacting with

chromatin at regulatory sites (promoters, enhancers)

possess, along with DNA-binding and effector domains,

a conserved RNA-binding domain resembling the argi-

nine-rich motif (ARM) of the HIV-1 Tat protein [123].

Integrated analysis of the DNA–protein interactions

(ChIP-seq) and RNA–protein interactomes (CLIP-seq) of

several transcription factors (GATA1, YY1, and CTCF)

revealed interactions of these factors with the RNA

originating from the loci near the factor binding sites

on chromatin (enhancer, promoter, and nascent tran-

scripts). Mutations in the ARM-like domain led to the

decreased expression of target genes, while its deletion

increased proportion of the freely diffusing transcrip-

tion factors in the nucleus. These findings suggest that

interactions of transcription factors with the nascent

RNAs via the ARM-like domain may contribute to for-

mation of the transcriptional condensates and provide

precise regulation of gene expression.

In line with the aforementioned observations, a

model for transcriptional condensate formation me-

diated by interaction of the nascent RNAs and protein

factors possessing RNA-binding domains and unstruc-

tured regions that promote formation of biomolecu-

lar condensates (RNA-mediated feedback model) ap-

pears attractive [122, 124]. In the experiments utilizing

in vitro and in silico systems, it was demonstrated that

the RNA molecules, both from promoters or enhancers,

irrespective of their sequence, promoted formation

of transcriptional condensates involving the Media-

tor complex [125]. However, this effect was observed

only within a limited range of RNA concentrations,

with the fixed concentration of the Mediator complex.

At such RNA concentrations, the system reached an

equilibrium state between the negative charge of nu-

cleic acid, proportional to its length, and the positive

charge of protein factors. Increase in the amount of

RNA carrying a negative charge disrupted the equilib-

rium and led to the condensate dissolution. Validity

of the RNA-mediated feedback model proposed by the

authors was also demonstrated in the in vivo experi-

ments. Transcription initiation and production of the

short nascent transcripts led to formation of the tran-

scriptional condensate and simultaneous increase in

the reporter gene expression. However, when an ex-

cessive level of transcription and a threshold concen-

tration of the nascent RNAs were reached, expression

of the reporter gene decreased, and the temporarily

formed transcriptional condensates resulting from

nonspecific electrostatic interactions between the syn-

thesized transcripts and protein factors were dissolved

[124-126]. This mechanism of gene expression regula-

tion is realized in enhancers through the short-lived

enhancer RNAs [125, 126]. This mechanism can explain

transcriptional bursts observed for many genes in the

cases when the enhancer- and promoter-associated

condensates interact or fuse [126, 127].

CONCLUSION

The data accumulated in recent years indicate

that transcription is accompanied by formation of the

molecular complexes with non-stoichiometric composi-

tion – transcriptional condensates. Apparently, forma-

tion of such condensates is determined by combination

of the high-affinity specific interactions and nonspecif-

ic interactions between the proteins, RNA, and DNA

sites that are part of these condensates. Aswith other

described biomolecular condensates, transcriptional

condensates that contain RNAPII are extremely dy-

namic. In contrast, transcriptional condensates formed

as a result of RNAPI activity form stable membraneless

structures – nucleoli. Nevertheless, in many respects,

formation of the transcriptional condensates is similar

in the cases of RNAPI and RNAPII.

The IDRs of proteins engaged in transcription

play a pivotal role in formation of the transcription-

al condensates. Although these domains participate

in nonspecific interactions, they significantly enhance

efficiency and specificity of the condensate formation

during transcription. This applies to various process-

es, including both search for the target sites in the

nucleus and interaction between the individual com-

ponents. Notably, positive feedback mechanisms may

be at play, wherein molecular interactions lead to for-

mation of the transcriptional condensate facilitating

attraction of additional molecules of the same proteins

and potentially other molecules involved in the pro-

cess. This phenomenon is particularly evident in the

nucleolus, which attracts a diverse array of molecules

[80] contributing to its multifunctionality [128].

Nascent transcripts play a crucial role in the tran-

scription regulation. Transcripts still bound to poly-

merase or RNAs in the transcriptional condensate

due to the large negative charge of their sugar-phos-

phate backbone, can attract RNA-binding proteins to

the transcription site through nonspecific interactions

with the positively charged IDRs, along with specific

interactions facilitated by the RNA secondary struc-

tures. Varying length of RNA, ranging from a few tens

TRANSCRIPTION REGULATION AND TRANSCRIPTIONAL CONDENSATES 695

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

to several thousand nucleotides for different RNA

types, enables multivalent interactions with proteins.

Ability of the nascent transcripts to attract protein

factors to the transcription site can be used to de-

velop tools for gene expression regulation involving

CRISPR-Cas9 systems with activator domains of the

transcription factors [129].

However, it should be noted that many concepts

lack complete experimental validation. For example,

there is only limited data available on the structure of

elongation transcriptional condensates. Some morpho-

logical observations suggest that elongation of some

genes occurs on the extended DNA strands resembling

loops of the lampbrush chromosomes [35, 36]. While

these observations do not definitively contradict the

idea of the existence of elongation condensates, they

introduce some uncertainty. Moreover, consideration

should be given to the data on the transcriptional con-

densate dissolution promoted by high levels of the tran-

scribed RNA molecules [124-126]. Disassembly of such

condensate may facilitate movement of the RNAPII

molecules along the DNA strand. These questions re-

quire further investigation.

Thus, high efficiency and flexible regulation of

transcription largely stem from the weak, non-specif-

ic, yet multiple interactions that give rise to formation

of the highly dynamic at the molecular level transcrip-

tional condensates.

Contributions. A.A.V. and E.V.Sh. collected and an-

alyzed information, wrote and edited the manuscript.

Funding. The work was financially supported

by the Russian Science Foundation (project no. 21-74-

20134).

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

Theauthors of this work declare that they have nocon-

flicts of interest.

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