Chromosome Res (2023) 31:8
https://doi.org/10.1007/s10577-023-09714-y
REVIEW
Nuclear architecture and the structural basis of mitotic
memory
Mamilla Soujanya · Ashish Bihani · Nikhil Hajirnis · Rashmi U. Pathak · Rakesh K. Mishra
Received: 5 July 2022 / Revised: 13 November 2022 / Accepted: 19 December 2022
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract The nucleus is a complex organelle that
hosts the genome and is essential for vital processes
like DNA replication, DNA repair, transcription, and
splicing. The genome is non-randomly organized
in the three-dimensional space of the nucleus. This
functional sub-compartmentalization was thought
to be organized on the framework of nuclear matrix
(NuMat), a non-chromatin scaffold that functions as
a substratum for various molecular processes of the
nucleus. More recently, nuclear bodies or membrane-
less subcompartments of the nucleus are thought to
arise due to phase separation of chromatin, RNA, and
proteins. The nuclear architecture is an amalgamation
of the relative organization of chromatin, epigenetic
landscape, the nuclear bodies, and the nucleoskeleton
Responsible Editor: Helder Maiato
M. Soujanya · A. Bihani · N. Hajirnis · R. U. Pathak ·
R. K. Mishra
CSIR - Centre for Cellular & Molecular Biology,
Hyderabad, India
M. Soujanya · R. K. Mishra
AcSIR - Academy of Scientific and Innovative Research,
Ghaziabad, India
N. Hajirnis
Department of Anatomy and Neurobiology, University
of Maryland, Baltimore, USA
R. K. Mishra (*)
TIGS - Tata Institute for Genetics and Society, Bangalore,
India
e-mail: mishra@ccmb.res.in
in the three-dimensional space of the nucleus. Dur-
ing mitosis, the nucleus undergoes drastic changes
in morphology to the degree that it ceases to exist
as such; various nuclear components, including the
envelope that defines the nucleus, disintegrate, and
the chromatin acquires mitosis-specific epigenetic
marks and condenses to form chromosome. Upon
mitotic exit, chromosomes are decondensed, re-estab-
lish hierarchical genome organization, and regain
epigenetic and transcriptional status similar to that of
the mother cell. How this mitotic memory is inher-
ited during cell division remains a puzzle. NuMat
components that are a part of the mitotic chromosome
in the form of mitotic chromosome scaffold (MiCS)
could potentially be the seeds that guide the relative
re-establishment of the epigenome, chromosome ter-
ritories, and the nuclear bodies. Here, we synthesize
the advances towards understanding cellular memory
of nuclear architecture across mitosis and propose a
hypothesis that a subset of NuMat proteome essential
for nucleation of various nuclear bodies are retained
in MiCS to serve as seeds of mitotic memory, thus
ensuring the daughter cells re-establish the com-
plex status of nuclear architecture similar to that of
the mother cells, thereby maintaining the pre-mitotic
transcriptional status.
Keywords Nuclear architecture · Mitotic memory ·
Transcriptional memory · Ribonucleoprotein
network · Nuclear matrix (NuMat) · Mitotic
chromosome scaffold (MiCS) · Mitotic
bookmarking · Phase separation · Nuclear bodies
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Abbreviations
hnRNP Heterogenous nuclear ribonucleoprotein
LAD Lamina-associated domains
MiCS Mitotic chromosome scaffolds
NuMat Nuclear matrix
NOR Nucleolar-organizing region
SAF-A Scaffold attachment factor A
SAF-B Scaffold attachment factor B
S/MARs Scaffold/matrix-associated regions
TAD Topologically associated domains
Introduction
In eukaryotes, the nucleus is the largest and most
prominent organelle in the cell that hosts the genome
and has to be faithfully replicated into daughter cells
(Cavalier-Smith 1988). The linear DNA is wrapped
around histone octomers as nucleosomes to form
the chromatin. It is regulated by several epigenetic
mechanisms like DNA methylation, post-translational
modifications of the histone tails, and non-coding
RNA determined by DNA sequence specificity (Mis-
teli 2007). In an organism, the same genome sequence
is differentially organized in the three-dimensional
space of the nucleus, and the epigenetic landscape
is differentially modified to give rise to distinct cell
type-specific transcriptional outputs (Lanctôt et al.
2007; Golbabapour et al. 2011). The transcriptional
status of a cell eventually dictates its identity and
function as the outcome of differentiation process
(Schneider and Grosschedl 2007; Guo 2014). Differ-
entiated cells must pass their epigenetic and transcrip-
tional memory to the daughter cells without aberra-
tions to retain their identity and function (Soufi and
Dalton 2016).
The interphase nucleus is a congregation of several
dynamic processes like transcription, RNA process-
ing, and DNA repair, which are shown to be halted
to a major extent during mitosis (Prescott and Bender
1962; Liang et al. 2015). In the interphase nucleus,
chromatin interacts with several phase-separated
membrane-less nuclear bodies like transcription fac-
tories, DNA replication foci, Cajal bodies, nucleolus,
and polycomb bodies, to carry out different functions
(Dundr and Misteli 2010a; Mao et al. 2011; Banani
et al. 2017). Activity-dependent nuclear bodies are
assembled over chromatin and are locus- and cell
type-specific. The three-dimensional organization of
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chromatin and its epigenetic landscape is crucial for
interacting with these nuclear bodies (Dundr 2012;
Wood et al. 2014; Koch 2018). Some nuclear bod-
ies undergo disassembly during mitosis and rapid
re-assembly upon mitotic exit, whereas some per-
sist through mitosis (Dundr and Misteli 2010a). The
mechanisms regulating this process are shown to be
synchronized with the cell cycle kinase–phosphatase
activities (Rai et al. 2018). Nuclear architecture and
function are co-dependent properties that emerge
due to the relative interactions between the chroma-
tin, nuclear bodies organizing in a chromatin-specific
context, and the underlying nucleoskeleton that could
tether and relatively position various nuclear bodies
and chromatin with respect to each other. However,
the mechanisms driving the accurate re-assembly of
nuclear bodies in the daughter nucleus and memory
for their association with the self-organizing chroma-
tin and nuclear architecture are yet to be understood.
Cell division is a complex process during which
the cell undergoes several physiological and mor-
phological changes. In preparation for mitosis, the
genome is duplicated, during which histones and
other proteins bound to DNA are dislodged and the
DNA double helix is unwound for replication. Fur-
ther, the nuclear envelope is disrupted, chromosome
territories are lost, chromatin is condensed into the
chromosomes, and nuclear bodies are disassembled
(Woodcock and Ghosh 2010; Boettcher and Barral
2013; Kutay et al. 2021). There has been increasing
evidence that the transcriptional status, epigenetic
landscape, and three-dimensional organization of the
chromatin are promptly re-established post-mitotic
exit. Thus, the two daughter cells have the same func-
tional state as the mother cell (Gerlich et al. 2003;
Shoaib et al. 2020). During decondensation of the
chromosome post-mitosis, the chromosome territo-
ries are re-established. The hierarchical organization
of the genome from compartments to topologically
associated domains (TADs) and loops is precisely
organized, and the genome is functionally compart-
mentalized into several nuclear bodies in the correct
chromatin context. The inactive TAD at the nuclear
envelope interacting with the nuclear lamina is clas-
sified as lamina-associated domains (LADs) (Guelen
et al. 2008) and comprise 30–40% of the genome
(Vogel et al. 2007; Peric-Hupkes et al. 2010). LADs
that are common to many cell types and gene-poor
are known as constitutive LADs and that differ
Chromosome Res (2023) 31:8
between cell types and comprise of clusters of devel-
opmentally important genes are which called faculta-
tive LADs (Peric-Hupkes et al. 2010). Reorganization
of LADs is shown to be necessary for differentiation
(Guelen et al. 2008; Peric-Hupkes et al. 2010; Kohwi
et al. 2013). It has been shown that movement of
LADs from periphery to interior or vice versa hap-
pens during mitosis (Harr et al. 2015); however, very
little is known about how the LADs are precisely re-
established post-mitosis. During mitosis, the lamins
are phosphorylated in a cell cycle-regulated manner
and disassembled with the nuclear envelope (Ottavi-
ano and Gerace 1985); simultaneously, the chromatin
is detached from the periphery and condensed into
chromosomes. Lamin C remains associated with the
LADs on mitotic chromosomes and correlates tempo-
rally and spatially with the post-mitotic nuclear enve-
lope association of LADs (Wong et al. 2021). Lamins
are abundantly present and are a major contribu-
tor to the filamentous organization of both NuMat
and MiCS (He et al. 1990; Nickerson et al. 1997).
Attachment of the genome to the nucleoskeleton
occurs through scaffold/matrix attachment regions
(S/MARs), and these regions have been shown to be
associated with lamins (Ludérus et al. 1992; Zhao
et al. 1996). However, there is limited understanding
of the role of NuMat and MiCS in the re-establish-
ment of LADs. Although they appear to be individual
and independent steps, they all occur synchronously
and are interdependent. Nuclear matrix could poten-
tially bridge and coordinate interphase nuclear archi-
tecture processes (Meier et al. 2005). It is enigmatic
how the nucleus undergoes these drastic changes dur-
ing mitosis yet re-establishes the spatial aspects of
nuclear architecture and gene regulation with remark-
able precision (Lodhi et al. 2016) (Fig. 1).
Over the decades, several models have been pro-
posed for the mechanism of “mitotic memory” trans-
mission. It could be in the form of mitotic chromo-
some-specific epigenetic modifications (Schmitz et al.
2020), chromatin-associated proteins, and RNAs
which are essential for marking and maintaining the
gene regulatory landscapes during chromatin conden-
sation (Bellec et al. 2018). One of the models is based
on the epigenetic status of the chromatin at the mitotic
chromosome that allows the transcription factors to
bind to their targets in the telophase and re-estab-
lish coordinated gene expression (Zaidi et al. 2011;
Kadauke and Blobel 2013). Histone post-translational
Page 3 of 23 8
modifications (PTMs) have been associated with dif-
ferent functional states of the genome (Goldberg
et al. 2007; Gibney and Nolan 2010). Histone PTMs
differentially mark the genome of a cell into broadly
categorized euchromatin and heterochromatin regions
in a context-specific manner (Bannister and Kou-
zarides 2011). There is a steady gain of knowledge
eliciting phase separation as the driver of euchroma-
tin and heterochromatin genome organization depend-
ent on the histone modifications (Strom et al. 2017;
Larson and Narlikar 2018; Falk et al. 2019). Histone
post-translational modifications have been associ-
ated with several stages of mitosis and are shown to
be indispensable for proper chromatin condensation
and segregation (Wang and Higgins 2013; Wilkins
et al. 2014; Schmitz et al. 2020; Halsall et al. 2021).
The most dynamic and mitosis-specific modification
is phosphorylation at residues such as H3S10ph and
H3S28ph across the chromosome, H3T3ph at inner
centromeres, and H3T11ph at centromeres (Loomis
et al. 2009; Doenecke 2014; Alabert et al. 2015; Biggs
et al. 2019). These modifications are essential to facili-
tate the evacuation of HP1 and RNA pol II from chro-
matin (Margueron and Reinberg 2010). Methylation
marking the transcriptionally active regions such as
H3K4me2 and H3k4me3 is restricted to the 5′ end of
the genes. Heterochromatin or repressive methylation
such as H3K9me2, H3K9me3, and polycomb-medi-
ated H3K27me3 is maintained across mitosis (Wang
and Higgins 2013). H3K9me2 is shown to mark
LADs and, during mitosis, acquires the dual modifi-
cation H3K9me2S10ph to inherit the spatial position-
ing of LADs at the lamin or periphery of the nucleus
(Poleshko et al. 2019). Acetylation associated with
active transcription is globally reduced during mitosis,
and persistent acetylation is essential for post-mitosis
transcription activation (Behera et al. 2019). Histone
variants such as H3.3 and H2AZ are chromatin loci-
specific and essential for mitosis (Ng and Gurdon
2008). The dynamics of several other post-transla-
tional modifications and variants of histones are yet to
be understood in the context of chromatin condensa-
tion and decondensation during mitosis.
Another model known as the “mitotic book-
marking model” posits that the transcription fac-
tors remain bound to the chromosomes, acting as
“mitotic bookmarks” through the cell cycle (Kadauke
and Blobel 2013; Zaret 2014; Palozola et al. 2019).
Although several transcription factors are shed from
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Fig. 1  A subset of NuMat is retained in MiCs as a form of
mitotic memory. A Representation of an interphase nucleus. B
During prophase, the nuclear membrane begins to disintegrate,
and the chromatin condenses to form mitotic chromosomes.
C Highly condensed metaphase chromosomes. D Treatment
of interphase nucleus by high salt buffers and DNAse ren-
ders a meshwork of non-histone proteins, RNA, and residual
DNA and is called the nuclear matrix (NuMat). A subset of the
the chromatin during chromatin condensation and are
synthesized upon mitotic exit, several proteins that are
a part of the transcriptional machinery, such as RNA
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NuMat are the proteins essential for mitotic memory. E Rep-
resentation of data from Sureka et al. (2018), LC/MS analy-
sis of NuMat, and MiCS show 67% overlap in the proteins
(highlighted in blue). F Treatment of mitotic chromosomes
with high salt buffers and DNase renders MiCS. MiCS retains
a major subset of NuMat proteins, which are essential for re-
establishing the interphase nucleus upon mitotic exit
pol I, RNA pol II, splicing speckles protein SC35,
steroid hormone receptors, and chromatin remodel-
lers, are retained on the mitotic chromosome (Zaidi
Chromosome Res (2023) 31:8
et al. 2010). Several sequence-specific transcrip-
tion factors such as Sox2, PARP1, GATA1, Fox A1,
and Brd4 occupy specific loci in the genome across
the cell cycle (Deluz et al. 2016; Teves et al. 2018;
Behera et al. 2019). These locus-specific interactions
of transcription factors enable rapid reinstatement of
gene expression of the associated genes post-mitosis,
thus acting as “mitotic bookmarks.”
Multiclassifier combinatorial proteomics has ena-
bled the identification of the majority of proteins
constituting the mitotic chromosomes that were previ-
ously unknown (Ohta et al. 2010). 3D correlative light
and electron microscopy (3D-CLEM) has provided
evidence that chromatin is not the major component
of the mitotic chromosomes (Booth et al. 2016).
Understanding the whole proteome of the mitotic
chromosome has provided essential insights into the
diversity of the proteins constituting the chromo-
some. Probing the functions of proteins differentially
enriched spatially on the chromosome could provide
clues to decipher the principles of spatial and struc-
tural recapitulation of nuclear architecture. While
some of these proteins are present on the surface of
the chromosomes, some are directly associated with
the chromatin in the loops of the mitotic chromo-
some and others at the mitotic chromosome scaf-
fold (MiCS) (Booth et al. 2014). Nonetheless, there
is very little or no evidence for the significance of
their association with mitotic chromosomes. A recent
model argues that most genes maintain the micro-
environment required for transcription even in the
highly condensed chromatin state of mitotic chromo-
somes which re-establishes the transcriptional status
upon mitotic exit (Palozola et al. 2019). Though sev-
eral proteins essential for processes like DNA replica-
tion, DNA repair, and transcription, are known to be
present on the mitotic chromosome (Ohta et al. 2010),
we understand very little about whether proteins and
RNA bind to the chromatin to facilitate the conden-
sation process, bookmark the transcriptional status of
the genes, or simply hitchhike on chromosomes for
transportation in appropriate proportions or all of the
above. As yet, no model explains the re-establishment
of the macro-environments or the nuclear architec-
ture of the interphase nucleus. The nitty–gritty of
mitotic chromosome organization, post-mitotic reor-
ganization, and the unplumbed role of RNAs and
proteins are yet to be understood. Understanding the
composition of interphase chromatin and the mitotic
Page 5 of 23 8
chromosomes is essential to decipher the mode and
means of transport of the architectural, spatial, and
temporal aspects of mitotic memory across cellular
states in the cell cycle.
A classical model for the functioning of the inter-
phase nucleus obligates the presence of nuclear
matrix (NuMat) a non-chromatin nucleoskeleton
that functions as a tethering ground for transcription,
DNA replication, and DNA repair (Getzenberg 1994;
Berezney and Wei 1998). The components of NuMat
are subjected to cell cycle regulatory mechanisms,
and the function of NuMat across the cell cycle
phases has remained speculatory (Loidl and Eberhar-
ter 1996). NuMat and MiCS proteomes are a subset
of the nuclear proteomes at different cell cycle phases
with similar protein compositions, which may be far
from mere similarity. They could provide a core set
of proteins that need to be probed further to dissect
the principles of nuclear architecture and the struc-
tural basis of mitotic memory. Comparison of NuMat
and MiCS biochemical constituents suggest the pos-
sibility that NuMat components packaged into mitotic
chromosomes in the form of MiCS are essential for
transmission of nuclear architecture through mitosis
(Fig. 1) (Sureka et al. 2018).
Nuclear matrix and mitotic chromosome scaffold
The structure and organization of the nucleus capti-
vated researchers’ attention right from when it was
deemed a container of hereditary material by Ernst
Haeckel (Haeckel 1988, 1904). Early attempts to
examine the nuclear architecture were through bio-
chemical solubilization of the loosely bound com-
ponents followed by imaging. One of these treat-
ments, the sequential extraction of the nucleus with
0.4 M and 2 M NaCl, could solubilize the chroma-
tin, while some components remained attached to
the nucleus (Mirsky and Pollister 1942, 1946). This
helped uncover an underlying ribonucleoproteina-
ceous network — which was also confirmed via
visualizations of RNP bodies in situ (Smetana et al.
1963; Monneron et al. 1972). To better visualize the
architecture, the solubilized chromatin was removed
using nucleases, leading to compaction of the RNP
network and manifesting in the form of a filamentous
network (Zbarskiĭ 1990; Berezney and Coffey 1974;
Verheijen et al. 1988). This morphologically striking
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structure of NuMat not only explained the reason for
the mechanical robustness of the nucleus but also
provided a model for the subnuclear organization
(He et al. 1990). Research over the years has shown
transcription and replication to occur in hubs local-
ized on NuMat, in contrast with the previous under-
standing of homogenous and diffused distribution
across the nucleus (Wilson 1899; Jenke et al. 2004;
Hnilica 2018). Observations of DNA repair, splicing,
and RNA processing associated with nuclear matrix
consolidated the idea that nuclear matrix served as a
scaffold and substratum to subnuclear enzymatic pro-
cesses (Berezney and Wei 1998).
The NuMat proteome has been characterized
using mass spectroscopy. It consists of a subset of
the nuclear proteome comprising structural proteins,
chaperones, DNA/RNA binding proteins, chroma-
tin remodelers, transcription factors, etc. (Kallappa-
goudar et al. 2010). Most NuMat proteins are essen-
tial for the organization and compartmentalization
of the genome and consist of several proteins essen-
tial for seeding nuclear bodies (Sureka et al. 2018).
Further, comparing NuMat proteomes from different
developmental stages of Drosophila melanogaster
showed that less than half of the NuMat is conserved
across cell types. The rest of the proteins are stage-
specific dynamic components (Varma and Mishra
2011). NuMat has also been shown to be cell type-
specific and displays variability in the constituent pro-
teins between different cell types (Puri et al. 2021).
A whole proteome comparative analysis of NuMat
of several organisms have shown that the major cat-
egories of proteins remain the same (Calikowski et al.
2003; Kallappagoudar et al. 2010; Varma and Mishra
2011; Engelke et al. 2014). A subset of the NuMat
proteome remains associated with the mitotic chro-
mosomes, implicating their role in mitotic memory
(Kallappagoudar et al. 2010).
Akin to the nucleus, when the mitotic chromosomes
are subjected to biochemical treatments with high salt
buffers and nucleases, the histones are depleted, and a
largely proteinaceous MiCS is uncovered (Adolph et al.
1977). Several early studies, based on HeLa cells, pro-
posed that NuMat and MiCS comprise similar proteins
(Detke and Keller 1982). Nevertheless, the identity
of these proteins remained unknown until an in-depth
proteomic study was carried out to identify the com-
ponents of NuMat and MiCS in Drosophila S2 cells.
According to this data, 67% of proteins are common to
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NuMat and MiCS, 27% are unique to NuMat, and 6%
are unique to MiCS (Fig. 1E) (Sureka et al. 2018). Thus,
MiCS contains seeds as points for the continuation of
the interphase state after mitosis that are essential for
the spatial organization of the genome and compartmen-
talization into several nuclear bodies. Similar catego-
ries of proteins were observed as constituents of MiCS
derived from DT40 chicken cells (Ohta et al. 2019). The
dispersed network of NuMat in the interphase nucleus
transitions to a condensed scaffolding structure in the
mitotic chromosome (MiCS) during mitosis. The pro-
teins common between NuMat and MiCS are heat shock
proteins, RNA binding proteins, replication-associated
proteins, rRNA-associated proteins, histone modifiers,
and mediator complex proteins. The constituents of
NuMat and MiCS provide a promising set of candidates
from different subcompartments of the nucleus that can
be probed further to better understand the reminiscence
of several molecular processes like transcription, DNA
replication, and repair on the mitotic chromosomes
in the form of mitotic memory and their roles in re-
establishment of these processes after mitosis. Retain-
ing these complexes on the mitotic chromosomes might
also be responsible for maintaining microenvironments.
These microenvironments are essential for processes
like transcription leading to low levels of transcriptional
activity that have recently been shown to occur during
mitosis (Timmers and Verrijzer 2017).
We hypothesize that a subset of NuMat compo-
nents is packaged/retained into mitotic chromosomes
in the form of MiCS. Components of MiCS with
retention of epigenetic marks and transcription factor
bookmarking could integratively create the micro-
environment of the gene to transmit mitotic memory
during cell division. MiCS proteins function as poten-
tial seeds that provide a form of structural mitotic
memory preserved during mitosis and are essential
for re-establishing a functional interphase nuclear
architecture by coordinating the establishment of the
spatial organization of the genome via phase separa-
tion of chromatin and nuclear bodies.
NuMat proteins are essential for genome
organization across the cell cycle
Chromosomes in the interphase nucleus occupy
specific niches in the three-dimensional space of
the nucleus relative to each other, known as the
Chromosome Res (2023) 31:8
chromosomal territories (Cremer et al. 2001).
Advancements in high-throughput chromosome con-
formation capture assays and super-resolution micro-
scopes have led to a refined understanding of the
genome. The genome is hierarchically organized into
transcriptionally active or euchromatic compartment
A and transcriptionally repressive or heterochromatic
compartment B (Lieberman-Aiden et al. 2009; Rao
et al. 2014). Genome organization is speculated to be
driven by the interplay of loop extrusion and phase
separation (Conte et al. 2022). There is accumulat-
ing evidence eliciting the role of phase separation in
heterochromatin and euchromatin formation, which
is reflected in compartments A and B of the genome
(Strom et al. 2017; Larson and Narlikar 2018; Falk
et al. 2019; Singh and Newman 2020). Meso- and
microphase separation arising from lamina associa-
tion and transcription has also been linked to spatial
chromatin organization (Hilbert et al. 2021; Bajpai
et al. 2021). Polymer modeling using principles from
soft matter physics provides a theoretical understand-
ing of the mechanism of phase separation driving
genome organization (Nuebler et al. 2018; Bajpai
et al. 2021; Laghmach et al. 2021; Brahmachari et al.
2022; Fujishiro and Sasai 2022). Phase separation has
also been implicated as a mechanism orchestrating
several mitotic structures and functions (Hernandez-
Verdun 2011; Booth et al. 2014; Brugués and Needle-
man 2014; Jiang et al. 2015; Woodruff et al. 2017;
Huang et al. 2017; Rai et al. 2018; Park et al. 2019;
King and Petry 2020; Ong and Torres 2020; Sten-
ström et al. 2020). Accumulating evidence also sug-
gests that hyper-deacetylation of chromatin, coating of
chromosomes by Ki-67 render a phase transition that
regulates organization and structure of mitotic chro-
mosomes (Cuylen et al. 2016; Schneider et al. 2022).
The euchromatic compartment A and heterochro-
matic compartment B are further divided into sub-
compartments that are collections of self-interacting
topologically associated domains with similar transcrip-
tional properties (Sexton et al. 2012; Dixon et al. 2012;
Nora et al. 2012). TADs have been shown to create
microenvironments at the megabase level to facilitate
necessary interactions and insulate them from the rest
of the genome to prevent cross-regulation of genes lead-
ing to misexpression. Recently, reorganization of TADs
to promote cell fate transitions was shown to be depend-
ent on phase separation of OCT4, disrupting which
attenuated TAD reorganization during reprogramming
Page 7 of 23 8
(Wang et al. 2021, p. 4). The TADs are further organ-
ized as a set of chromatin loops. The chromatin loops
arise due to the interaction of regulatory elements with
genes such as enhancer–promoter interactions and are
transcriptionally relevant (Rao et al. 2014). There are
several architectural proteins essential for the organi-
zation of the genome at different levels, and the most
studied are the proteins involved in the loop extrusion
mechanism (Guillou et al. 2010; Phillips-Cremins et al.
2013; Van Bortle et al. 2014; Ganji et al. 2018). The
mammalian genome is majorly organized by the insu-
lator protein CTCF along with cohesin via the DNA
loop extrusion mechanism and has been implicated in
mitotic memory (Klenova et al. 1993; Birshtein 2012;
Shen et al. 2015). In Drosophila, several other insula-
tor proteins, such as Su(Hw), CP190, GAF, BEAF32,
and dCTCF, are essential for genome organization, and
deletion of dCTCF had minimal effect (Kaushal et al.
2021). Some of these insulator proteins involved in
DNA looping and genome organization have recently
been shown to undergo phase separation to form insula-
tor bodies along with loop extrusion component Rad21
(Amankwaa et al. 2022). Phase separation emerges as a
conserved mechanism for genome organization across
evolution (Feric and Misteli 2021). However, phase
separation and genome organization have been stud-
ied in a locus-specific manner. They lack explanations
for the trafficking of proteins and RNA across several
nuclear bodies for their processing. During chromatin
condensation, the locus-specific chromatin interactions
are lost, and chromatin loop rosettes are organized lon-
gitudinally on a linear axis (Oomen and Dekker 2017;
Miura and Hiratani 2022). To re-establish the compart-
ments, TADs, and specific interactions essential for
gene regulation, proteins bookmark the features of the
3D genome and epigenome along with the re-establish-
ment of processes like transcription, RNA processing,
and replication. Several architectural proteins and struc-
tural maintenance of chromosome proteins are essential
components of the interphase and mitotic chromosome
organization and are constituents of both NuMat and
MiCS (Fig. 2).
Insulator binding proteins in genome organization
Insulator proteins play a crucial role in inter-
phase chromatin organization and gene regulation
(Gaszner and Felsenfeld 2006; Dorman et al. 2007).
The binding of insulator proteins characterizes the
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Fig. 2  Proteins essential for genome organization are retained
on NuMat and MiCS. Representation of an interphase nucleus
and a chromosome, architectural proteins or insulator binding
factors, and structural maintenance of chromosome complexes
TAD boundaries to demarcate the TADs, block
enhancer–promoter interactions, and prevent the
encroachment of epigenetic modifications (Matharu
and Ahanger 2015). In Drosophila, several pro-
teins like BEAF32, GAF, Mod(Mdg4), Su(Hw), and
CP190 are known to function as insulators (Vogel-
mann et al. 2014). BEAF32, along with DREF and
others, has persisted on the mitotic chromosome at
sites highly enriched for Orc2 and Mcm2 at TAD
borders (Gurudatta et al. 2013). It is speculated that
DREF and insulator proteins bookmark sites for the
assembly of pre-replication complexes and gene
bookmarking during the mitosis to interphase tran-
sition (Gurudatta et al. 2013). Similarly, GAF is an
insulator protein that is essential for transcriptional
memory by stable bookmarking of genes like scyl in
Drosophila (Bellec et al. 2022). Transcription fac-
tor Myc has been shown to remain associated with
mitotic chromosomes colocalized with insulator pro-
teins (Yang et al. 2013). dCTCF partially remains
associated with mitotic chromosomes. However, its
consequences in the maintenance and propagation
of genome architecture across the cell cycles must
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— condensin and cohesin complexes. These proteins are
essential for chromosome assembly and could also perform the
function of mitotic memory upon mitotic exit and bring about
genome organization similar to the mother cell
be investigated (Shen et al. 2015). Several of these
insulator proteins (BEAF32, CP190, Mod(Mdg4),
Su(Hw)) are retained as MiCS from NuMat (Fig. 2).
Understanding the cell cycle dynamics of these archi-
tectural proteins is essential to decoding mitotic book-
marking and the principles of genome organization.
Cohesins and condensins in genome organization
In the interphase nucleus, cis-based DNA looping is
required for communication between distal regulatory
elements like enhancers and promoters. Cohesins and
CTCF have been shown to organize the interphase
genome via loop extrusion essential for gene regula-
tion, organizing replication foci (Guillou et al. 2010;
Yan et al. 2013). After DNA replication, the sister
chromatids are held together by cohesin complexes.
These complexes undergo degradation at the meta-
phase-to-anaphase transition for cell cycle progres-
sion. Several components of the cohesin-mediated
mitotic chromosome folding complexes like Tra1,
SA, and San are constituents of both NuMat and
MiCS (Valdeolmillos et al. 2004; Sureka et al. 2018).
Chromosome Res (2023) 31:8
Their presence in MiCS may not be strictly restricted
to their mitotic function and requires further probing.
Similarly, condensins are multi-subunit protein
complexes that play a central role in mitotic chromo-
some assembly and segregation (Hirano 2012). Con-
densins also play an important role in homolog pair-
ing and polytene disassembly in Drosophila, gene
regulation, maintenance of chromosome territories,
DNA damage repair, and cell type-specific functions
in the interphase nucleus (Wallace and Bosco 2013).
Upon nuclear envelope breakdown, chromatin con-
densation in prophase is orchestrated by condensins
II and I SMC proteins by hierarchical folding or suc-
cessive chromatin coiling from the chromosomes’
peripheral region, subsequently leading to the forma-
tion of stable mitotic chromosomes with resolved sis-
ter chromatids (Hirota et al. 2004; Batty and Gerlich
2019). Maeshima and Laemilli propose a different
model in which topoisomerase II mediates the early
stages of compaction followed by the condensins
(Maeshima and Laemmli 2003). The condensin com-
plex and topoisomerase II proteins are present in both
NuMat and MiCS. The sequence of events at which
these proteins are essential for mitotic chromosome
assembly is still elusive.
Though it is apparent to see several of these pro-
teins as a part of MiCS, understanding the cell cycle
resolved dynamics of these proteins is essential to gain
insights into the mechanisms of genome compaction
and decompaction. Investigating the role of subunits
of cohesin and condensin complexes in mitotic book-
marking is required to understand the principles of 3D
genome folding, packaging, and in particular the re-
establishment of all this after mitosis (Fig. 2).
MiCS: the miniaturized NuMat as mitotic
memory of nuclear bodies and functions
Upon mitotic exit, decondensed chromatin needs to
be re-organized in the three-dimensional space of the
nucleus such that the chromatin is compartmental-
ized, like the mother cell (Lodhi et al. 2016, p.). A
large proportion of interphase nucleus and mitotic
chromosomes is made up of proteins, indicating the
crucial role of the protein environment in shaping the
chromatin landscape (Booth et al. 2016). The massive
qualitative overlap between the two, as estimated by
proteomics of NuMat and MiCS, shows that ~ 67% of
Page 9 of 23 8
the NuMat environment is transmitted unperturbed
through the complex process of mitosis via MiCS
(Sureka et al. 2018). The interphase nucleus consists
of distinct nuclear bodies essential for replication,
transcription, DNA repair, splicing, and ribosome
subunit assembly (Mao et al. 2011). During mito-
sis, these nuclear bodies undergo partial or complete
disassembly and reassemble upon interphase re-
entry (Dundr and Misteli 2010a). We discuss below
proteins that are core components of nuclear bodies
present in NuMat which are packaged as MiCS in
the context of phase separation and nuclear bodies
biogenesis.
Nucleolus The most starkly visible example of
nuclear bodies is the nucleolus. This complex mem-
brane-less organelle forms around clusters of rDNA
genes (nucleolar-organizing regions (NORs)) and
mainly serves as a compartment for the transcription
and assembly of ribosomal subunits (Bártová et al.
2010). The nucleoli disassemble as the chromosomes
condense, and their components disperse during pro-
phase and reassemble during interphase. Several
nucleolar proteins have been shown to have non-
canonical functions during mitosis (Hernandez-Ver-
dun 2011; Fujimura et al. 2020). The nucleoli’s key
conserved structural constituents (Fibrillarin, Nop56,
and Nopp140) are abundant in the NuMat and remain
attached to MiCS (Sureka et al. 2018) contributing
to the proper coming together of NORs and forma-
tion of nucleolar subcompartments around them post-
mitosis. Mitotic chromosomes, in general, although
highly condensed, ribosomal genes, in particular,
are accessible to transcription factors and chromatin
remodelers (Chen et al. 2004). Re-establishment of
the nucleolus could be achieved by coordinated rRNA
transcription upon seeding by nucleolar proteins of
NuMat packaged into MiCS (Fig. 3).
Nuclear speckles Nuclear speckles contain the pre-
mRNA processing factors and non-coding RNAs, and
they work in concert to coordinate multiple steps of
gene expression, including pre-mRNA processing,
storage, transport, and recycling of splicing factors
(Galganski et al. 2017). SR protein SF2 is a common
component of the NuMat and MiCS and is associated
with interphase chromatin, released from the hyper-
phosphorylated chromosomes but reassociated with
chromatin late in M-phase to recruit HP1 (Loomis
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Fig. 3  Proteins essential for nuclear bodies in NuMat are
retained as seeds in the MiCS. Representation of an interphase
nucleus with nuclear bodies represented in different colors.
Proteins from these nuclear bodies are a part of NuMat in the
interphase cell, and they undergo disassembly during mitosis
et al. 2009). SF2 depleted cells released from mitotic
block displayed delayed G0/G1 entry, suggesting a
functional consequence of these interactions (Loomis
et al. 2009). The nuclear speckle re-assembly depends
on the reactivation of transcription at late telophase
and could be nucleated by the nuclear speckle pro-
teins from NuMat that are present in MiCS (Fig. 3).
Omega speckles Stress during mitosis results in
chromosome missegregation, aneuploidy, or apop-
tosis unless mitigated (Burgess et al. 2014). Heat
shock factors are transcriptional regulators of heat
shock proteins in the interphase nucleus and have also
been implicated in mitigating stress in mitotic chro-
mosomes despite global inhibition of transcription
during mitosis (Vihervaara et al. 2013; Elsing et al.
2014). In Drosophila, HRB87F are essential com-
ponents of nuclear stress bodies and are present in
the NuMat and MiCS (Biamonti and Vourc’h 2010;
Sureka et al. 2018). These proteins facilitate the coun-
teraction of transcriptional silencing, protect cells
from proteotoxicity during mitosis, and potentially
serve as mitotic memory components from NuMat
that are packaged in MiCS for the re-establishment
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and are retained on MiCS in the mitotic chromosomes. These
proteins in MiCS are potential candidates for mitotic memory
and act as seeds of nucleation for the re-assembly of nuclear
bodies in the same genomic context in the daughter cells upon
mitotic exit
of nuclear stress bodies or omega speckles (Fig. 3)
(Vihervaara et al. 2013; Elsing et al. 2014; Sureka
et al. 2018).
Cajal bodies A closely associated nuclear body
with the HLB is the Cajal body, essential for transcrip-
tional regulation of histone genes and snRNA genes
(Dundr and Misteli 2010a). Coilin is a Cajal body
marker protein that is also shown to be associated with
HLB in Drosophila (Ogg and Lamond 2002). During
mitosis, Cajal bodies are excluded from the vicinity
of the chromosomes and scattered in the cytoplasm.
Upon nuclear envelope reformation, Coilin rapidly
re-enters the nucleus (Dundr and Misteli 2010b).
Recently, Cajal bodies were also linked to genome
organization at sn/snoRNA, small CB-specific RNA
(scaRNA), and histone gene loci. Cajal body proteins
tethering to these loci during mitosis are essential for
bookmarking the context of its re-assembly. Coilin is a
component of NuMat and is retained in MiCS (Sureka
et al. 2018); hence, further investigation is essential
to understand the Coilin phosphorylation dynamics
during the cell cycle and biogenesis of Cajal bodies
(Fig. 3) (Hearst et al. 2009).
Chromosome Res (2023) 31:8
Polycomb bodies Polycomb group of proteins are
known as the epigenetic regulators of transcription
and are implicated in cellular memory to remember
the active and repressed transcription states over sev-
eral cell divisions. The binding of PcG proteins to
establish the repressive domains leads to an increase
in their concentration on chromatin loci and the for-
mation of polycomb bodies (Müller and Verrijzer
2009; Cheutin and Cavalli 2012). Several loci are
clustered to co-regulate at the polycomb bodies (Pir-
rotta and Li 2012). Polycomb is retained in MiCS
from the NuMat (Sureka et al. 2018) as a nuclea-
tion site for polycomb body reformation at the book-
marked loci after cell division (Fig. 3).
Replication foci The early and late replicating
regions of the genome are cell type-specific and must
be marked across mitosis (Grasser et al. 2008; Ryba
et al. 2011). Some of NuMat/MiCS member proteins
like Orc2, Orc5, and Mcm2 are well-known players
in replication, and these proteins are essential for
mitotic chromosome assembly by recruiting kinesins
to disassemble the replication foci, and mutations in
these proteins have been implicated in chromosome
condensation and chromatin organization defects
(Pflumm and Botchan 2001). These replication foci
bookmarking by Orc1 have also been shown to be
essential for the spatio-temporal re-establishment of
DNA replication similar to the mother cell (Fig. 3)
(Kara et al. 2015).
Polycomb group proteins Polycomb group pro-
teins form several complexes that serve as epige-
netic transcriptional repressors through their ability
to read and write histone modifications (Margueron
and Reinberg 2011). While the core PRC1 complex
(Psc, Sce) can write H2A-K119-ubiquitin marks, the
PRC2 core (E(z), Su(z)12, Esc, Caf1-55) has H3K27
di- and tri-methylation activity — responsible for lay-
ing down facultative and constitutive heterochromatin
states, respectively (Margueron and Reinberg 2011;
Schuettengruber et al. 2017). PhoRC (Sfmbt, Pho)
binds H3K9me1 and H3K20me2 and consolidates the
scaffold of polycomb bodies (Klymenko et al. 2006;
Seif et al. 2020).
Among the members of polycomb groups com-
plexes, Pc, Sce, Psc, E(z), Su(z)12, Ph, and Pho are
retained on S2 cell mitotic chromosomes (Follmer
et al. 2012). Another report shows that E(z) and Pho
Page 11 of 23 8
dislodge from Drosophila embryo (stage 13–15)
mitotic chromosomes, and Pc is found in smaller
amounts — which implies that some of these pro-
teins are consistently associated with mitotic chro-
mosomes (Follmer et al. 2012; Steffen et al. 2013). A
comparison of polycomb complex occupancy across
cell cycle shows that the binding sites in mitosis are
a small subset of the sites in interphase, implying
that these overlapping sites signify the bookmarking
sites (Follmer et al. 2012). Pc interacts with SMC1/3
(Strübbe et al. 2011), which are also part of both
NuMat and MiCS structural scaffold components
for different nuclear processes. We observe that Pc
(PRC1/2), Sfmbt (PhoRC), and Caf1-55 (PRC2) are
common between NuMat and MiCS.
Another point in the cell cycle that has been stud-
ied in the context of mitotic memory is the S-phase,
where, during the dilution of epigenetic marks
through replication, certain loci appear to replicate
the epigenetic moieties using environments created
by PcG/TrxG and HP1 (Lanzuolo et al. 2011). This
replication has been stipulated to be on a broad region
instead of specific nucleosomes, which points to the
importance of PcG and HP1 compartments around
these loci. Zeste-mediated recruitment of Brm/Trx
onto specific sites in polytene chromosomes has also
been considered a possible mechanism of memory
transmission. In humans, CHMP1 is associated with
NuMat and is known to recruit PcG proteins for gene
silencing and affects chromatin structure and cell
cycle progression (Stauffer et al. 2001).
Trithorax group of proteins Trithorax group pro-
teins also form a diverse set of highly modular com-
plexes that function antagonistically to polycomb
complexes and maintain an epigenetically activating
influence on their target genes (Schuettengruber et al.
2017). Broadly, trithorax complexes are of two types,
BAF and COMPASS. Among BAF-type complexes,
canonical BAF, non-canonical BAF, and pBAF have
Bap55, Bap60, Mor, Act5C, Brd7-9, and Brm as
components. All COMPASS-type complexes (Set1/
COMPASS, Trr, and Trx) have Ash2, Rbbp5, and
Wds in common. Mutations in BRG1 (Dm Bap55)
and ARID1A (Dm Osa), in combination with Top2a,
have been associated with the proper disentangle-
ment of chromosomes during segregation in mitosis,
which is likely why BAF complex proteins are packed
with mitotic chromosomes (Dykhuizen et al. 2013).
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Ash2 only associates with NuMat and not MiCS,
while Wds is seen in both NuMat and MiCS. Trx is
known to associate with embryonic mitotic chromo-
somes but is not seen in S2 cell MiCS (L Black et al.
2016). Thus, COMPASS-type complexes are largely
absent from MiCS. On the other hand, proteins
associated with the Brahma/BAF complex (Bap55,
Bap60, Bap111, Bap170, Brm, Mor, Baf180/Poly-
bromo, Osa) are present in both NuMat and MiCS
(Sureka et al. 2018). This implies that BAF-type com-
plexes may be marking the active regions and utilize
their chromatin remodeler component to prepare for
transcription spike as the nucleus is formed rapidly.
However, the nature of the involvement of these
regions in maintaining mitotic memory needs further
investigation.
Transcription assembly The epicenter of recre-
ating a cell’s transcriptional landscape is the tran-
scription machinery, which is formed by a large
number of general transcription factors and RNA
polymerase subunits. Recruiting, releasing, and
pausing this complex in the right loci are important.
This function of bookmarking and memory storage
by RNA Pol II complex has been confirmed (Mich-
elotti et al. 1997). If the occupancy of RNA Pol II
subunits on mitotic chromosomes is disrupted, the
transcriptional landscape of daughter cells is heav-
ily affected (Teves et al. 2018). Two main units of
this complex, Polr2A and Polr2D, are present in
both NuMat and MiCS. Actively transcribed loci
are recruited for Condensin I loading, and inhibi-
tion of polymerase activity leads to reduced RNA
pol II and condensin I occupancy at these loci, con-
necting the RNA Pol marking of loci to the scaffold
(Fig. 3) (Sutani et al. 2015).
RISC complex proteins RNA-induced silenc-
ing complexes (RISCs) utilize small RNAs to target
and degrade various transcripts and are composed
primarily of Argonaute and Dicer family proteins.
Some RISC complexes also directly affect chromatin
organization, termed RNA-induced transcriptional
silencing complexes (RITSs). Small RNA-based tar-
geting leads to the recruitment of HP1 and HP2 (Pratt
and MacRae 2009). RISC has been implicated in pro-
moting epigenetic inheritance in C. elegans, which
requires the presence of polycomb and other chroma-
tin modifiers (Conine et al. 2013).
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Ago2 from RNA-RISC, a highly conserved pro-
tein across eukaryotes, remains associated with both
MiCS and NuMat, while Dcr-2 is present only in
NuMat. Ago2 has been genetically and biochemically
associated with telomeric and pericentromeric het-
erochromatin formation alongside Top3β — and the
absence of Ago2 causes the formation of anaphase
bridges (Lee et al. 2018) Ago2 has been implicated
in the formation of centromere and heavily overlaps
with HP1 and PcG occupation sites (Volpe et al.
2002). HP1a/b, HP4, HP5, and HP6 have also been
associated with chromosome inner centromere (Hig-
gins and Prendergast 2016) and are present in both
NuMat and MiCS (Sureka and Mishra 2018). Inter-
estingly, both PcG and RISC-based heterochromatin
compartments have reduced levels in mitotic chromo-
somes, which expand as the interphase sets in again
(Follmer et al. 2012).
Chromatin modifiers Several histone acetyltrans-
ferases, which activate transcription through H3
lysine acetylation, have also been seen to be retained
on both NuMat and MiCS, namely, Enok (Kat6a/b),
Nejire (P300), and Fs(1)h (Brd2-4) (Sureka et al.
2018). Another study found BRD4 (in a murine
GATA-/- cell line) being retained on mitotic chro-
mosomes in a subset of loci compared to interphase.
However, mitosis-specific inhibition of BRD2-4 bro-
modomain did not affect its recruitment to mitotic
chromosomes, indicating that these proteins are not
retained through their chromatin association activity
and could be mere passengers (Behera et al. 2019).
The mammalian homolog of Nejire (P300) is known
to be associated with the assembly of the transcrip-
tion pre-initiation complex (PIC) in conjunction with
mediator and condensin complexes (Wong et al.
2014). The knockout of P300 disrupts the transcrip-
tional program of several genes, and a defect in the
recruitment of cohesin and Brd4 is seen (Wong et al.
2014). However, further studies are needed to exam-
ine if the observed effect is indeed due to the disrup-
tion of mitotic retention of P300 only. The murine
homolog of HDAC1 has been implicated in locus-
specific retention of RBPJ in mitotic chromosomes of
F9 cells (Dreval et al. 2019) — and HDAC1, along-
side Mi-2 and Mep-1 of NuRD complex, is retained
in MiCS (Sureka et al. 2018). This complex is also
considered involved in the replication-based transmis-
sion of memory (Budhavarapu et al. 2013) (Fig. 3).
Chromosome Res (2023) 31:8
It is also likely that the RNA component of these
nuclear bodies is also retained in the mitotic chromo-
somes, given the presence of several non-coding and
intronic RNA that have been found to remain attached
to the chromosomes (Meng et al. 2016). Interestingly,
a large number of proteins of NuMat/MiCS are asso-
ciated with RNAs, not only belonging to transcript
processing but also to chromatin reorganization. For
example, scaffold attachment factor B (SAF-B), an
RNA binding protein, is known to be a part of the
mammalian NuMat and is involved in forming mem-
brane-less compartments. The interphase re-entry
is accompanied by multiple waves of transcription,
which, in coordination with the chromatin microen-
vironment seeded by the NuMat proteins in MiCS,
could result in specific interactions and the re-estab-
lishment of nuclear bodies at the bookmarked chro-
matin contexts (Palozola et al. 2017). The miniatur-
ized “seeds” of NuMat in the form of MiCS have to
expand then to re-establish the nuclear subcompart-
ments after mitotic exit. The proteomics datasets for
MiCS are only recent, and examining how this re-
expansion occurs requires candidate tracking based
experiments with multiple cell types in different
organisms to obtain a holistic picture.
During the post-mitotic re-establishment of the
compartmentalized nuclear architecture, the active
and repressive regions of the chromatin need to be
marked and targeted to distinct domains (Lodhi et al.
2016). Although the loop formation in mitotic chro-
mosomes is uniformly distributed across the genome,
the local organization of individual loci could be
affected by bookmarking and relative mobility of
transcription factors and epigenetics modules (Rac-
caud et al. 2019). Additionally, as the interphase chro-
matin landscape is a tug of war between inactivating
and activating chromatin-associated factors (Ghotbi
et al. 2021). The fairly common retention of these
factors on mitotic chromosomes could also reflect the
same and can be used to re-establish compartmentali-
zation and transcriptional activity upon the interphase
re-entry.
This genome scale effort cannot be executed by a
small set of factors, and many of the chromatin-asso-
ciated proteins would be involved. Many chromatin-
associated proteins are captured embedded in NuMat,
in a structural form and seem to be included in MiCS
(Sureka et al. 2018). Accordingly, a comparison
of the proteomes of NuMat and MiCS reveals that
Page 13 of 23 8
chromatin-modifying factors from the trithorax group
of proteins, polycomb from the polycomb group of
proteins, enzymes like histone acetyltransferases and
deacetylases, and RISC complex proteins which are
essential for the maintenance of heterochromatin are
found in both NuMat and MiCS, whereas histone
methyltransferases and demethylases are prominently
present in NuMat (Sureka et al. 2018) A large number
of these proteins are conserved across eukaryotes, and
their origin can be traced to bacterial and archaeal
lineages (Sureka and Mishra 2021). This implies
that these NuMat proteins are the evolutionarily con-
served core of the nucleus and serve key functions in
important nuclear processes like transcription.
The nucleic acids of NuMat and MiCS
Scaffold/matrix attachment regions
In the interphase nucleus, chromosomes are known to
occupy relative non-random positions in the nucleus
as chromosomal territories, which have been shown
to remain intact in NuMat (Ma et al. 1999). NuMat is
the scaffold over which chromatin loops are organized.
The DNA sequences that help attach the base of the
chromatin loops to the NuMat are defined as scaffold/
matrix-associated regions (S/MARs) and may repre-
sent the boundaries of individual chromatin domains.
Although there is not much sequence similarity in the
S/MARs, they are AT-rich and enriched in repetitive
DNA (de Belle et al. 1998; Arope et al. 2013; Pathak
et al. 2014). S/MARs are regulated by the S/MAR
binding proteins and their post-translational modifica-
tions (Bodnar et al. 1983). S/MARs have been shown
to interact with NuMat proteins such as SATB1 and
CDP/Cux for transcriptional regulation of CD8a genes
in mice. S/MARs have also been implicated in defining
the chromosomal breakage locations during oxidative
stress-induced apoptosis (Tan et al. 2018, p.). Several
locus-specific and genome-wide S/MARs have been
characterized over the years using techniques such as
MAR-seq and Matrix 3C (M3C) (Gavrilov et al. 2010;
Tan et al. 2018). Genome-wide mapping of MARs
shows that they are primarily the cis-regulatory ele-
ments of the genome and are enriched in gene-rich
regions in C. elegans and Drosophila melanogaster
(Anthony and Blaxter 2007; Pathak et al. 2014). In
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Bombyx mori, S/MARs have been shown to regulate
developmental stage-specific molecular mechanisms in
the nucleus (Chunduri et al. 2022). Comparing MARs
across normal vs. cancer cells and correlating it with
genome-wide epigenetic landscape and gene expres-
sion revealed that the MARs in normal cells comprise
actively transcribing genes, whereas MARs in cancer
cells comprised of repressed genes and were enriched
for chromatin modifications marking transcriptional
activation and repression, respectively. Thus, MARs are
speculated to be highly cell context-dependent (Dobson
et al. 2017). ChIP-seq of 14 S/MAR binding proteins
from the human NuMat have shed light on molecular
signatures of S/MARs and are implicated as hotspots
for integrating retroviruses (Narwade et al. 2019). S/
MARs have also found applications in minimizing the
effect of transgene silencing (Allen et al. 2000). Mini-
mal S/MARs are sufficient for replication and mitotic
stability of mammalian episomes via their interactions
with NuMat proteins such as SAF-A (Jenke et al. 2004).
The miniaturization of the NuMat to MiCS con-
cept would require that S/MARs are retained as
DNA loop attachment sites in MiCS. This was ini-
tially predicted and experimentally proved for the
first time by Laemmli’s group. They examined the
higher-order loop organization of DNA in interphase
nuclei and metaphase chromosomes to demon-
strate the conservation of DNA attachment regions
between these two cell cycle phases (Paulson and
Laemmli 1977). Further confirming a fundamental
role for these fragments in the organization of the
genome into looped domains, unpublished results
from our lab show that the S/MARs in interphase
are a subset of DNA attachment sites on the MiCS.
Due to the high compaction of chromatin in mitotic
chromosomes, many more DNA fragments are
retained on the MiCS compared to NuMat. In-depth
characterization of the S/MARs from NuMat and
MiCS will provide crucial insights into understand-
ing how interphase chromatin loops are packaged
on the mitotic chromosomes and their role in post-
mitotic chromatin reorganization.
RNA and RNBPs are an essential component of
nuclear architecture
RNA and RNA binding proteins play an important
role in nuclear architecture and spatial organization
of the chromatin in the nucleus (Nickerson 2022).
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RNA binding proteins such as heterogeneous nuclear
ribonucleoprotein (hnRNP) are abundant and prefer-
entially associated with NuMat (Gallinaro et al. 1983;
Fey et al. 1986). Non-coding RNA has been shown
to have several functions in spatial genome organiza-
tion of the peri-nuclear chromosome tethering, bio-
genesis of nuclear bodies, chromatin looping, and
maintenance of the epigenetic landscape (Khosraviani
et al. 2019; Latonen 2019; Nozawa and Gilbert 2019).
RNA–protein interactions act as molecular glue that
facilitates the formation of ribo-proteinaceous mesh
or NuMat that encompasses several nuclear bodies,
thus pointing to the suspected structural role of RNA
in genome organization (Hall and Lawrence 2016).
Scaffold attachment factors A and B are hnRNPU
proteins that are an abundant component of NuMat
and have emerged as important regulators of higher-
order nuclear organization and global maintenance
of 3D genome organization (Romig et al. 1992; Kipp
et al. 2000). Xist RNA causes condensation of the
inactive X chromosome into Barr body and NuMat-
associated proteins are essential for the organization
of the inactive X chromosome (Ridings-Figueroa
et al. 2017; Creamer and Lawrence 2017). SAF-A and
SAF-B are DNA-binding S/MAR-associated proteins
that are abundant in the inactive X chromosome and
are essential for the localization of Xist RNA (Kipp
et al. 2000; Helbig and Fackelmayer 2003; Hasegawa
et al. 2010). SAF-A/hnRNPU binds to tandem repeats
and S/MAR in a sequence non-specific but structure-
specific manner. SAF-A can bind to RNA and oli-
gomerize to form a homogenous mesh in an ATP-
dependent manner (Nozawa et al. 2017). SAF-A
also interacts with Cot1 repeat RNA and is essential
for interphase chromosome organization. Advance-
ments in super-resolution and live cell imaging tech-
niques have enabled the visualization of SAF-A mesh
(Marenda et al. 2022; Park et al. 2022). Non-coding
RNA have been proposed to have a role in the for-
mation of scaffold of RNP granules via phase sepa-
ration via the interaction between folded domains
and disordered low-complexity domains of hnRNPU
facilitate phase separation (Peng and Weber 2019;
Martin et al. 2021). SAF-A interacts several nuclear
RNA and SAF-B interacts with repeat elements RNA
to bring about phase separation to regulate chromatin
structure and transcription (Michieletto and Gilbert
2019; Huo et al. 2020). Depletion of SAF-B leads to
remodeling of 3D genome organization and global
Chromosome Res (2023) 31:8
SAF-A depletion which have been shown to increase
in LADs and redefinition of TAD boundaries (Fan
et al. 2018).
HNRNPUL1/SAF-A is a key component of Dros-
ophila melanogaster nuclear matrix but is not found
in MiCS (Sureka et al. 2018). It is also seen to be
evicted from the mammalian mitotic chromosomes
alongside a large number of RNAs (Kukalev et al.
2009; Sharp et al. 2020). On the other hand, SAF-B
localizes in transcription specific manner in the inter-
phase and remains associated with the mitotic chro-
mosomes (Alfonso-Parra and Maggert 2010; Sureka
et al. 2018). The family of serine-arginine-rich pro-
teins consists largely of nuclear proteins that bind to
RNA. A large number of these are involved in splic-
ing. The proteomic overlap between NuMat and
MiCS shows that several proteins of this family, like
B52 (SRSF4/6), SRp54 (SREK1), SC35 (SRSF2),
SF2 (SRSF1), and U2AFs, are retained on the mitotic
chromosomes (Sureka et al. 2018).
Most transcription is silenced and terminated in
mitosis, and the mitotic chromosomes are stripped of
the majority of transcripts (Sharp et al. 2020). Still,
a wide range of RNA types are found attached to
mitotic chromosomes in all phases of mitosis (Meng
et al. 2016; L Black et al. 2016). Mitotic chromosome
associated RNAs (mCARs) were identified using
5′-tag sequencing and comprised of several types of
non-coding RNA such as snoRNA, SINE RNA, and
uncharacterized novel RNAs (Meng et al. 2016). In a
recent study, the mammalian NuMat retained intronic
RNAs and lncRNAs, constituting a scaffold segre-
gated from chromatin domains alongside NuMat and
SAF-A (Creamer et al. 2021). In Drosophila mela-
nogaster, AAGAG repeat transcripts of several hun-
dred nucleotides are a critical component of NuMat
(Pathak et al. 2013). The integrity of NuMat depends
on ongoing transcription, implying that the nascent
transcripts have a role in building and maintaining
the nuclear architecture (Prescott and Bender 1962;
Poirier and Marko 2002; Tripathi et al. 2012). Inter-
estingly, non-coding, repeat-rich long nascent RNA
transcripts play a dynamic structural role in inter-
phase chromosome architecture (Creamer et al. 2021).
Though RNA is a significant component of NuMat,
very little is known about the RNA in MiCS. As dis-
cussed in the subsections about nucleolus, nuclear
bodies, and Cajal bodies, a large number of RNA
binding proteins members of NuMat are also retained
Page 15 of 23 8
in MiCS, which indicates a crucial role of RNAs in
re-establishing interphase nuclear architecture. While
the isolated MiCS is a structurally independent entity
that retains its metaphase morphology, RNA appears
to have a minimal role in its integrity. Exposure of the
chromosomes to a very high concentration of RNase
does not affect the isolation of histone-depleted
mitotic chromosomes (Adolph et al. 1977). Chromo-
some micromanipulation experiments also demon-
strate that the shape, structure, and mechanical prop-
erties of metaphase chromosomes depend primarily
on the integrity of RNA (Poirier and Marko 2002).
Since RNA plays a critical role in NuMat, under-
standing the RNA dynamics during mitosis will shed
light on the transcriptional kinetics through phases
of the cell cycle, its role in interphase nuclear archi-
tecture, and chromatin organization. Comparative
studies for NuMat/MiCS or nucleus/mitotic chromo-
some RNA need to be performed to understand the
structural transmission of RNAs across mitosis and
identify key transcripts that can potentiate mitotic
memory.
Conclusions
Cell division is mainly viewed as the segregation of
the genetic material equally to the daughter cells.
Along with the DNA, several proteins and modified
histones in the form of chromatin are also a part of
the mitotic chromosomes. The majority, if not all
of the MiCS, are NuMat proteins. These proteins
are essential for mitotic chromosome condensation,
bookmarking architectural features of chromatin
organization by insulator binding proteins that could
serve as potential anchor points for reorganization of
the decondensed chromatin in late telophase. Several
overlapping proteins between NuMat and MiCS are
epigenetic remodelers and regulators essential for
remodeling the epigenetic landscape during mitosis
and re-establishing the cell’s epigenome upon mitotic
exit. Nuclear bodies are indispensable components of
the nucleus and NuMat, which are hubs for carrying
out crucial steps of central dogma and maintaining
genome integrity and stability. Most nuclear bodies
are disassembled during mitosis and re-assembled
after cell division. The chromatin context in which
some of these bodies need to be built is carried in
MiCS as seeds for the biogenesis of nuclear bodies.
Vol.: (0123456789)
13
 8 Page 16 of 23
Using candidates from the proteomes of NuMat and
MiCS, canonical and non-canonical roles of several
proteins can be assessed to understand the mechanism
of mitotic memory. Similar to the NuMat, a subset of
S/MARs are also retained on MiCS, thus supporting
our hypothesis that NuMat proteome and S/MARs are
a form of mitotic memory. NuMat proteins, RNA and/
or S/MARs, have the potential to act as seeds to re-
establish the transcriptional status of the cell to pro-
vide its identity and function similar to their mother
cells. The in-depth understanding of the function of
S/MARs and RNA from NuMat and MiCS is largely
unexplored and yet to be characterized.
Acknowledgements The authors acknowledge the members
of the RKM lab and Shivakumara Manu for carefully reading
the manuscript and providing critical feedback.
Author contribution MS wrote and edited the manuscript
and created illustrations. AB wrote the review. NH wrote the
review. RUP wrote the review. RKM conceptualized and edited
the review.
Funding Research in the RKM lab is supported by grants
from the Council for Scientific and Industrial Research
(MLP0139), the Government of India, and the JC Bose fellow-
ship (GAP0466). MS is supported by DBT-SRF fellowship.
Data availability Not applicable.
Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests The authors declare no competing inter-
ests.
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