# The power of proximity: mechanisms and biological roles of transvection

Benjamin Prud'Homme

► To cite this version:

Benjamin Prud'Homme. The power of proximity: mechanisms and biological roles of transvection. Current Opinion in Genetics and Development, 2024, 89 (2), pp.273-281. 10.1016/j.gde.2024.102269. hal-04879706

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ELSEVIER

Review

# The power of proximity: mechanisms and biological roles of transvection

## Benjamin Prud'homme

<figure><img src="image_3.png" /></figure>

The phenomenon of transvection, defined as a proximity- dependent interallelic interaction, has been observed in the context of complementation between mutant alleles for numerous Drosophila genes. Cases of transvection-like phenomena have also been observed in other species, including mammals. However, the potential contribution of transvection to wild-type gene regulation and the underlying mechanisms remain uncertain. Here, I review recent evidence demonstrating the relevance of transvection in physiological contexts. These findings suggest that transvection represents an additional layer of gene regulation that allows cells to fine-tune gene expression based on the proximity of homologous alleles. In addition, recent studies have measured the physical distance between interacting alleles, revealing unexpectedly large and variable distances. I will discuss how these distances are compatible with the 'hub' model of transcriptional regulation.

### Address

Aix-Marseille Université, CNRS, IBDM, Institut de Biologie du Développement de Marseille, Campus de Luminy Case 907, Cedex 9, 13288 Marseille, France

Corresponding author: Prud'homme, Benjamin (benjamin.prudhomme@univ-amu.fr)

**Current Opinion in Genetics & Development** 2024, 89:102269

This review comes from a themed issue on **Genome Architecture and Expression**

Edited by Anders Sejr Hansen and Marcelo Nollmann

For complete overview of the section, please refer to the article collection, "Genome Architecture and Expression (2025)"

Available online 4 October 2024

https://doi.org/10.1016/j.gde.2024.102269

0959-437X/© 2024 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

The term transvection was introduced by Ed Lewis in 1954 to describe a specific type of genetic complementation between mutant alleles of the *Drosophila melanogaster* *Ultrabithorax* (*Ubx*) locus [1]. Complementation by transvection was later found to involve activation (or repression [2]) in trans of the promoter of a regulatory mutant allele by the enhancers of the homologous protein null allele [3–5]. In Drosophila, as in all

Diptera [6], the homologous chromosomes are paired, end-to-end, in all interphasic somatic cells. The pairing of the chromosomes is thought to facilitate transvection by keeping the homologous alleles relatively close together at all times [3,4]. Indeed, the complementation by transvection is prevented by chromosomal rearrangements that separate the *Ubx* alleles, suggesting that a close physical proximity of the two alleles is required for transvection to occur [1].

Following the pioneering work of Lewis, complementation by transvection between mutant alleles has been documented for several *Drosophila* genes [3,4]. In addition, some basic mechanistic aspects of transvection have been extensively studied using synthetic constructs [7,8]. While evidence for interchromosomal gene regulatory interactions exists in other species, including mammals, the number of cases is more limited [9].

Transvection sensu stricto has long been regarded as an unusual phenomenon, even by *Drosophila* geneticists, who have observed it only in certain allelic combinations. For many years, it remained unclear whether transvection played any role in gene regulation in a wild-type situation. However, recent studies have demonstrated the contribution of transvection to gene regulation in a variety of physiological and developmental contexts. In addition, these recent studies have begun to elucidate the underlying mechanisms of transvection, determining how the alleles come into close proximity and how this proximity can affect their regulation.

## Is transvection relevant for transcriptional regulation in wild-type, physiological contexts?

### Transvection averages out the allelic activities of autosomal loci

The *spineless* (*ss*) gene plays a critical role in determining the fate of photoreceptor cells in the *D. melanogaster* retina [10]. The expression pattern of *ss* in the retina is stochastic, occurring in a mosaic pattern in approximately two-thirds of the R7 photoreceptor cells. It is important to maintain this ratio, as it is critical for the development of color vision preferences [11]. The expression of *ss* is subject to a complex regulation during retinal development. Several enhancer and silencer cis-regulatory elements are involved at different stages of development [12]. In addition to their role on their cis-promoter, these regulatory sequences also
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

Transvection contributes to gene expression in a variety of wild-type contexts in Drosophila. (a) The ss locus is expressed in a mosaic pattern in the developing retina in approximately 66% of the R7 cells. This expression pattern results from the integration in cis of the activities of at least two enhancers (green boxes) and two silencers (red boxes) and from the averaging of the activities of these regulatory sequences between the two alleles (top). After deletion of one ss allele, the remaining allele is expressed in 54% of cells, a value that exceeds the expectations based on the assumption of additive regulatory activities of the two alleles (bottom). (b) In *D. biarmipes*, the X-linked gene *y* is expressed in a spot pattern in male pupal wings (top), whereas it is expressed in a salt-and-pepper pattern in a few cells in the female wing (middle). This sexual dimorphism is controlled by transvection, which, for yellow, can only occur in XX females. When one allele is deleted, *y* is expressed in a male-like spot pattern (bottom). (c) The gene *Stat92E* is expressed at high levels in a Drosophila male germline stem cell in the testis. In this cell, the *Stat92E* loci are more tightly paired than neighboring loci. In differentiating daughter cells of this stem cell, the expression level of *Stat92E* is reduced by about half compared to the stem cells, and the *Stat92E* alleles are more distantly located than in the stem cells (top). Deletion of one *Stat92E* allele does not alter the pairing state of *Stat92E*, and its expression level remains constant (bottom). This suggests that a change in local pairing can affect transcriptional regulation.

Adapted from Antel et al., 2022

interact in trans (Figure 1a), provided that they are in close
proximity to each other [13]. Consequently, the interaction
between the two alleles averages out the activating and re-
pressing inputs of each allele [12]. This averaging interaction
serves to limit the variation in *ss* expression, thereby con-
tributing to the maintenance of a finely tuned color detec-
tion system.

A similar role for transvection in regulating natural variation
in expression has recently been described for the *Malic
enzyme (Men)* gene [14], which encodes an enzyme required
for the reduction of the cofactor Nicotinamide Adenosine
Dinucleotide Phosphate+ to Nicotinamide Adenosine Di-
nucleotide Phosphate.

The regulation of *ss* and *Men* exemplifies the buffering
effect of transvection, which compensates for differences
in allelic activity, thereby maintaining gene expression
levels within a certain range and ensuring a robust
phenotypic output in the face of genetic variability or
developmental perturbations.

Transvection of an X-linked gene controls its sexually dimorphic expression

Unlike autosomal genes such as *ss* or *Men*, which are
always present in two copies in somatic cells, X-linked
genes are present in two copies in XX females but in a

single copy in XY males. This imbalance in the
number of X chromosomes between the sexes pro-
vides an opportunity for female-specific regulation of
X-linked genes through transvection [15], resulting in
sexually dimorphic transcriptional activity. While the
majority of sexually dimorphic gene regulation, in-
cluding that of X-linked genes, is controlled by the
sex-determining gene hierarchy [16], a first case of
sexual dimorphism at a transvecting X-linked gene has
recently been identified (Figure 1b). In *D. biarmipes*, a
species closely related to *D. melanogaster*, males display
a pigmentation spot on the wing tip that is partly
specified by the sexually dimorphic expression pattern
of the *yellow* (*y*) gene [17,18]. The sex-specific tran-
scriptional regulation of *y* results from an interaction
between the *y* alleles that silences *y* transcription in
the wing spot region [19]. Since *y* is X-linked, this
interaction occurs only in XX females and not in XY
males. Interestingly, this interallelic interaction is not
exclusive to the X chromosome or to the females. In-
deed, it is observed in both sexes using transgenes
inserted on the autosomes of *D. melanogaster*. This
example demonstrates how transvection can exploit
the inherent imbalance of the heterogametic chromo-
some between the sexes to induce sexually dimorphic
gene expression.
## Local changes in chromosome pairing and gene regulation

It is conceivable that transvection is more common in the genus Drosophila (and Diptera in general) than in other lineages because homologous chromosomes are paired in all somatic cells. However, the pattern of pairing can vary locally [20,21], at a particular stage or in a particular cell type, leading to changes in trans-regulation. An example of this is the regulation of the *Stat92E* gene during male germline differentiation in *D. melanogaster* (Figure 1c). *Stat92E* is expressed at high levels in the germline stem cells, where the *Stat92E* alleles are tightly paired. However, in the differentiating daughter cells, the pairing at the *Stat92E* locus is loosened, accompanied by a rapid decline in *Stat92E* expression levels [22]. This local change in pairing state appears to be required for the subsequent downregulation of *Stat92E* and the initiation of cell differentiation. This example illustrates how local and transient variation in chromosome pairing, a process that can be genetically regulated [23–26], can introduce an additional structural layer to the control of gene regulation.

The preceding examples show that transvection contributes to gene regulation in a variety of biological contexts in Drosophila and suggest that more examples can be found. They illustrate how trans-regulatory interactions provide an additional layer of gene regulation, allowing cells to fine-tune gene expression based on the proximity and regulatory activity of homologous alleles. A fundamental question about transvection is therefore how the proximity between the two alleles is regulated.

## How do alleles and chromosomes find each other in the nucleus?

Trans-regulatory interactions in Drosophila are often disrupted by genomic rearrangements, such as translocations or inversions, which increase the genomic distance between alleles [3]. This further supports the close relationship between pairing and transvection. However, it is important to stress that pairing and transvection are distinct concepts. The term ‘pairing’ is a subjective term used in the literature to describe the proximity of alleles (but how close is close?). In contrast, the term ‘transvection’ has a clear functional definition, referring to a regulatory interaction between alleles. It is crucial to distinguish these concepts, to identify the molecular determinants that influence chromosomal or allelic pairing (if they are different), and to assess how they might affect the strength of transvection.

### Sequence-specific or non sequence-specific ‘button’ models of chromosome pairing

Early studies of pairing initiation during *Drosophila* development suggested that regions of high pairing affinity (or ‘buttons’) are distributed along chromosomes, gradually bringing the homologous chromosomes closer together [27,28]. Recent live-imaging analyses in the

early embryo support a button-based mechanism for the initiation of somatic homolog pairing [29]. However, the molecular nature of these buttons remains unclear. A number of studies have shown that small DNA fragments containing insulator sequences can pair at considerable distances or even between chromosomes [30,31]. More recently, it has been shown that DNA segments containing complete topologically associated domains (TADs), including their borders enriched for insulator sequences, are more likely to mediate pairing than incomplete TADs [32]. However, it is clear that not all complete TADs can promote pairing, and conversely, pairing regions do not always overlap with complete TADs. This suggests that our understanding of the role of these domains in homolog pairing is still incomplete. Recent studies have shown that the TAD boundaries determine the formation of TADs [33,34], suggesting that the same sequences may control the cis-organization of chromatin along a chromosome as well as the trans interaction between homologs [25,35].

An alternative model to the sequence-specific button model has recently been proposed [36] (Figure 2c). This model, which is independent of TAD organization, proposes that specific spatial arrangements of short segments containing nonspecific association domains form a unique barcode pattern that organizes the pairing between homologs. Future experimental work should test and compare these models and investigate why certain buttons have a higher pairing capacity than others. It will also be important to relate to the nature of the DNA buttons to the various factors that have been shown to regulate chromosome pairing [23,26].

Whatever the exact nature of the buttons that pair homologous chromosomes, it is clear that the degree of pairing can vary. The proportion of cells in which chromosomes are defined as ‘paired’ is never 100% [27,28], and structural studies have shown that some regions of the *Drosophila* genome are ‘loosely’ paired, while others are more ‘tightly’ connected [20,21]. Although these concepts are difficult to translate into variations in the physical distance between alleles, all available data indicate that the strength of pairing is not uniform along chromosomes, varies between cell types and stages, and that this variation in local pairing can contribute to changes in trans-regulatory interactions [37–41], as illustrated above for the gene *Stat92E* [22]. It is therefore essential to understand how variation in pairing intensity translates into variation in physical distances and potentially interallelic gene regulation.

## Measuring interallelic distances: How close is close?

The fundamental principle of transvection is that homologous alleles must be in close proximity to interact
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>

Button models of homologous chromosome pairing. (a,b) The TAD — pairing button model postulates that the button sequences that organize the pairing of homologous chromosomes in fruit flies correspond to regions that span the TADs. (a) The buttons may span the entire TAD or discrete sequences within the TAD (double arrows). (b) Alternatively, the buttons may correspond to the TAD boundaries (colored ovals), which are enriched for insulator sequences and may in some cases promote self-pairing. (c) The nonspecific button barcode model assumes that short segments containing nonspecific association domains (orange bars) are arranged in different barcode patterns, which together form specific buttons (yellow rectangles). Regardless of the pairing model, the strength of the buttons can vary between different cell types, or along the chromosome, resulting in variable interallelic distances. These distance variations can locally influence the strength of transvection.

functionally. Surprisingly, the distributions of physical distances separating homologous loci have rarely been measured or compared between cells at different developmental stages [28, 42]. Measuring the distances between homologous loci is important for inferring the possible molecular mechanisms of transvection. This includes determining whether the distances are consistent with direct physical interactions between DNA molecules or between protein complexes bound to them. An alternative hypothesis is that the observed distances are more compatible with a larger (~200–300 nm) shared environment in which transcriptional regulators can

diffuse and act in a confined space, regulating both alleles simultaneously.

One limitation of measuring distances between paired alleles in *Drosophila* using classical confocal microscopy is the optical resolution, which is limited by diffraction. Using a single DNA Fluorescent In Situ Hybridization probe to label the two alleles can only resolve interallelic distances greater than 250–300 nm. To measure shorter distances, it is essential to distinguish between the two alleles. This can be achieved either by designing allele-specific DNA FISH probes based on sequence polymorphism [43] or by labeling each endogenous allele with a unique sequence [42], and using two-color localization microscopy. Application of this approach to fixed samples at the *Ubx* locus, which is known to transvect [2], revealed unexpectedly large median distances between transcriptionally active alleles and substantial variation between cells [42]. Furthermore, significant discrepancies in median distances were observed when measurements were performed at the *Ubx* promoter (~235 nm) or at an enhancer of *Ubx* located 45 kb away (~345 nm). This suggests that the interallelic distance can vary even within a single locus.

The observed variability in interallelic distance measurements can be attributed either to fixed differences between cells or to temporal fluctuations. Results from live-imaging experiments of transvecting synthetic constructs in the *Drosophila* embryo provide support for the latter hypothesis: the median interallelic distance is approximately 280 nm, with a relatively large standard deviation of ~120 nm [44]. Although further measurements of interallelic distances across different loci are clearly needed, these results indicate that interacting alleles can communicate over a considerable distance and that this distance is not fixed but rather fluctuates substantially and rapidly. This raises further questions about the mechanisms by which alleles can functionally interact at such distances and the extent to which variation in distance along the chromosome or between cell types and stages serves as a regulatory mechanism for interallelic interactions.

## 1+1≠2: how does transvection affect gene regulation?

What are the consequences for transcriptional regulation when two alleles are in close proximity to interact? Many studies of transvection have shown that an enhancer of one allele can activate transcription from the promoter of the homologous allele. It is therefore pertinent to ask whether they are able to communicate at the measured distances of approximately 250 nm. This distance is within the range of distances measured between active enhancers and their target cis-promoters in *Drosophila* and mouse embryos (median of 200–300 nm) [45–48]. It
is possible that these distances are overestimated due to
technical limitations and approximations. Nevertheless,
the observation that the distances between interacting
alleles are not significantly different from the distances
between coupled enhancer/promoter pairs suggests that
interactions in cis or in trans rely on similar physical and
organizational properties. Similarly, the observed varia-
tion in distance (in cis or in trans) suggests that both
modes of regulation are subject to the same fluctuation
dynamics.

These observations are consistent with the transcrip-
tional ‘hub’ model, which postulates that all the reg-
ulatory sequences of a locus share a common 3D space
that is also occupied by the large transcription initiation
complex [49,50]. This occupancy is thought to preclude
close contact between functionally interacting regulatory
elements and the promoter in the cells in which the
gene is transcribed. In line with this model, two paired
alleles would be in close proximity, forming a tran-
scriptional ‘trans hub’ [8,44]. The alleles could then in-
teract in this shared regulatory space, either competing
for shared transcriptional resources or co-operating (e.g.
through cooperative recruitment of transcription factors)
to generate novel interallelic-specific regulatory effects
(Figure 3). In this view, the regulatory elements of two
the alleles could interact with each other in a manner

analogous to the interactions observed between different
cis-regulatory sequences, including competition [51] or
co-operation. The proximity of the two alleles could also
influence the structure, stability, composition, and tem-
poral dynamics of the hub.

Perspectives

Many regions of the Drosophila genome have been
shown to support transvection, and transvection in mu-
tant contexts has been demonstrated for a large number
of genes. Surprisingly, however, the contribution of
transvection in physiological contexts, between wild-
type alleles, has only recently been established for a
modest number of genes. This raises the question of
whether this reflects a difficulty in detecting transvec-
tion for the majority of wild-type genes or whether this
phenomenon is relatively limited to a few genes. Further
studies will clarify the prevalence of transvection.

The available data suggest that interallelic distances can vary between cell types and stages. This suggests that the degree or strength, and possibly the temporal dynamics, of pairing may be regulated to influence gene expression. It will be interesting to investigate whether and to what extent transvection is involved in activating (or repressing) gene expression, which may rely on a transient interallelic proximity, versus maintaining a transcriptional state, which may require a prolonged interallelic interaction.

The phenomenon of interchromosomal gene regulation
has been documented in a number of species, including
vertebrates. In contrast to the situation in Drosophila,
homologous chromosomes are not paired in vertebrate
somatic cells [52]. Nevertheless, local and transient
chromosomal encounters can occur, which in some cases
have been observed to correlate with changes in gene
expression, both in wild-type and disease contexts [9,
53–56]. Such encounters may involve homologous loci or
genes located on non homologous chromosomes. Al-
though most of these examples are correlative, they sug-
gest that interchromosomal gene regulation may also
occur in vertebrates [57]. Only a comprehensive experi-
mental investigation can test the functionality of these
interactions [58]. In any case, the mechanisms underlying
interchromosomal communication in vertebrates remain
poorly understood, with only a few exceptions [59]. It is
plausible that these cases of interchromosomal gene reg-
ulation are transvection-like phenomena that share some
general regulatory principles. The extent of their simi-
larity will only be revealed by a detailed analysis of the
underlying molecular mechanisms.

Data Availability

No data were used for the research described in the ar-
ticle.

<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

The trans-hub model of transvection. (a) Each allele alone may not have the capacity to trigger transcription from a given enhancer (green rectangle). (b) In contrast, the physical proximity of the two alleles, including their regulatory regions, creates a microenvironment that concentrates regulatory proteins, favors regulatory interactions, and recruits the RNA polymerase II complex and components of the general transcriptional machinery to initiate transcription. This trans-hub model extends the classical transcriptional hub model, in which distinct cis-regulatory elements coalesce in the same microenvironment to regulate transcription.
# Declaration of Competing Interest

No conflict of interest.

# Acknowledgements

I thank the members of my lab for discussions on the topics discussed in this review. This work is supported by the Fondation pour la Recherche Médicale (EQU202103012586), the Agence Nationale de la Recherche, and Centre National de la Recherche Scientifique.

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