TICB 1746 No.

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Trends in
Cell Biology
Forum
and genetic database of individuals with and adult brain tissue, but the dynamic pro-
Chromatin dynamics global developmental delay, intellectual cesses that underlie brain organogenesis
in human brain disability, and microcephaly, among other are still inaccessible [6]. Advances in stem
development and developmental conditions, are involved in cell technologies have enabled the genera-
chromatin regulation. These genes include tion of neural cell types and circuits in
disease a member of the mammalian SWI/SNF organoids and assembloids (Figure 1). Inte-
(mSWI/SNF or BAF) chromatin remodel- grating these with single-cell resolution map-
Alfredo M. Valencia 1,2 and
ing complex, ARID1B; a member of the ping holds great potential for understanding
Sergiu P. Pașca 1,2,*
Mediator complex involved in transcrip- neurodevelopment and uncovering the
tional activation, MED13L; the transcrip- mechanisms underlying chromatinopathies.
tional repressors MECP2 and ANKRD11;
Chromatin-related genes are fre-
the histone modifiers KMT2A and CREBBP; Characterizing chromatin activity
quently mutated in neurode- and the mSWI/SNF interacting transcription and regulation
velopmental disorders; yet, the factor ADNP [3]. This underscores the Single-cell sequencing, including RNA and
mechanisms by which these pertur- importance of investigating how mutations chromatin accessibility profiling, is begin-
bations disrupt brain assembly and in these genes impact neural develop- ning to disentangle the heterogeneity of
function are not understood. Here, ment and give rise to disorders collec- complex tissues, including cell diversity
we describe how recent advances tively known as ‘chromatinopathies.’ The across human brain regions and over the
in transcriptional and chromatin pro- importance of studying these conditions course of development. These studies, per-
filing in combination with cellular using patient-derived cells is further formed in primary tissue samples, are now
models are beginning to inform highlighted by the fact that disruption of complemented by studies using organoids,
our understanding of neurodevelop- different chromatin genes results in similar which recapitulate some of the cell diversity
clinical phenotypes or that mutations in in the nervous system and can, in long-term
ment and chromatinopathies.
the same or a related chromatin gene can culture, advance to a postnatal signature
result in distinct syndromes. For example, [7]. Recent evidence demonstrates highly
Kleefstra syndrome, which is characterized dynamic, cell-specific chromatin changes
Chromatin and chromatinopathies by ID, hypotonia, and epilepsy, was initially during corticogenesis [8]. Further studying
Eukaryotic genomes are packaged into a associated with mutations in the histone the distribution of chromatin regulatory
highly ordered structure called ‘chromatin’, methyltransferase EHMT1. However, muta- proteins and histone marks during brain
which is regulated by a diverse set of pro- tions in a different histone methyltransferase, development could reveal the cell type–
teins that work collaboratively to regulate KMT2C (MLL3); the mSWI/SNF chromatin specific regulatory landscape.
gene expression. Chromatin is dynamically remodeling gene SMARCB1 (INI1); the
modulated by proteins that epigenetically DNA methyl binding protein MBD5; or the The most widely used approach for char-
modify DNA and histones, as well as protein transcription factor NR1I3 can also cause acterizing chromatin modifications and
complexes that physically alter genomic Kleefstra syndrome [4]. Furthermore, muta- chromatin binding proteins is ChIP-seq.
accessibility and topology. Coordination tions in genes encoding members of the For ChIP-seq, cells are fixed using an
of chromatin activity in time and space is mSWI/SNF chromatin remodeling com- agent to covalently crosslink chromatin-
essential for fundamental cellular processes plex, including SMARCB1 and ARID1B, bound proteins to DNA. Chromatin is
as well as for multicellular events, such among others, are implicated in ASD, non- then sheared, target DNA bound pro-
as organogenesis. Remarkably, chromatin syndromic ID, and syndromic NDDs Coffin- teins are immunoprecipitated, and isolated
regulatory genes are among the most fre- Siris syndrome (CSS) and Nicolaides- DNA is sequenced. ChIP-seq experiments
quently mutated genes in neurodevelop- Baraitser syndrome [5]. typically have low signal-to-noise ratio, re-
mental disorders (NDDs), including autism quiring significant sequencing reads and
spectrum disorder (ASD), intellectual dis- Given that chromatinopathy-related genes many cells. However, two recently devel-
ability (ID), and epilepsy [1,2]; yet, the span a broad range of processes, under- oped chromatin mapping methods –
mechanisms underlying their pathogene- standing genome regulation, especially in CUT&RUN and CUT&Tag [9] – allow the
sis remain elusive. the context of human brain development, characterization of histone modifications
is critical. Multiple recent efforts have started and chromatin binding proteins similarly to
Seven of the top ten genes with the most to characterize the transcriptional and chro- ChIP-seq. They offer advantages over tra-
sequence variants in DECIPHER, a clinical matin accessibility profiles of human fetal ditional ChIP-seq, including ease of use,

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Trends in Cell Biology

Figure 1. Functional genomic approaches and human cellular models to investigate the transcriptional and chromatin regulatory landscape of the
human brain. Human cellular models of brain development and disease. Somatic cells, including fibroblasts or peripheral blood mononuclear cells, can be isolated
and reprogrammed into human induced pluripotent stem cells (hiPSCs), which can then be aggregated and differentiated using instructive signals into self-organizing
tridimensional cultures known as brain region–specific organoids. Integration of multiple organoids into assembloids allows modeling of more complex cellular
interactions, including neuronal migration and neural circuit formation. Functional genomics. Organoids and assembloids can be dissociated for single-cell
transcriptional analysis (scRNA-seq) to reveal cell diversity over differentiation. Chromatin accessibility profiling by assay for transposase-accessible chromatin with
high-throughput sequencing (ATAC-seq) provides a broad portrait of genomic regions that are open and accessible. Targeted chromatin mapping techniques, such as
CUT&RUN, CUT&Tag, or ChIP-seq, can be used to examine histone post-translational marks, transcription factors, or chromatin binding proteins. Last, DNA
methylation can be examined through bisulfite sequencing or microarray methods and provide insights into the dynamics of gene regulation in neural differentiation.

increased sensitivity, and tractability in a Given the high prevalence of chromatin and assembloids will allow to comprehen-
single-cell format. For these methods, regulatory gene perturbations in NDDs sively profile the regulation, topology, and
cells or nuclei are immobilized and perme- and the fact that disorders can arise from transcriptional activity of the genome dur-
abilized on a solid support, and then anti- mutations at every level of chromatin ing neurodifferentiation and in disease.
bodies targeting the protein of interest are organization (Figure 2), mapping ap-
added. Either MNase endo-exonuclease proaches to uncover chromatin marks Cellular defects in chromatinopathies
(CUT&RUN) or Tn5 transposase (CUT&Tag) and chromatin binding proteins in a Human stem cell–based models have
fused to protein A/G is subsequently used genome-wide manner could inform devel- begun to reveal some of the neuronal and
to selectively cleave and solubilize target- opmental processes and therapeutic inter- glial phenotypes of NDDs. Mutations in
bound DNA. The inherent cellular diversity ventions. To date, only a few studies have MeCP2, an epigenetic reader that binds
of neural tissue and organoid models used single-cell CUT&RUN or CUT&Tag to methylated DNA to promote transcrip-
makes single-cell profiling techniques in- to study neural tissues [10]. Implementing tional repression, have been implicated in
credibly valuable in parsing out cell type– these methods more broadly in patient- Rett syndrome, an NDD characterized by
specific chromatin signatures. derived 2D cultures and 3D organoids loss of motor skills and speech that starts

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Trends in Cell Biology

in the first year of life. Interestingly, almost

all missense mutations reside within the
methyl-binding domains of the protein that
mediate transcriptional repression, pointing
to the domain-specific activity disrupted in
this disorder. Cortical organoids derived
from individuals with Rett syndrome were
smaller in size than in controls, had lower
expression of synaptic genes, and demon-
strated a reduction in presynaptic puncta
[11]. Future work characterizing the localiza-
tion of MeCP2 across the genome over
longer periods of differentiation and in
more disease-relevant brain regions could
inform future therapies.

The ARID1B gene, which encodes a

component of the mSWI/SNF chromatin
remodeling complex, is one of the most
commonly mutated genes in ASD and non-
syndromic ID [1,12]. A variety of heterozy-
gous truncation mutations of ARID1B have
been identified in nonsyndromic cases of
ID with agenesis of the corpus callosum.
Furthermore, ARID1B is the most frequently
mutated gene in CSS, which is charac-
terized by ID, coarse facial features, and
hypotonia [12]. Intriguingly, the mutually
exclusive ARID1B paralog, ARID1A, is
among the most frequently mutated
genes in cancer. Not much is known
about the distinct roles that ARID1A
and ARID1B play in chromatin regulation.
Using ARID1B+/– patient-derived hiPSCs,
a recent preprint discovered a key
switch between ARID1B containing BAF
(ARID1B-BAF) and ARID1A-BAF that can
influence exit from a pluripotency state
[13]. Failure to make this switch was related
to aberrant binding of SOX2 and impaired
cranial neural crest differentiation in individ-
uals with CSS [13]. Exploration of how
these mutations and others impact the
generation of cell diversity in the cerebral Trends in Cell Biology
cortex would be valuable in understanding
Figure 2. Chromatin organization and chromatinopathy-related genes. Neurodevelopmental disorders
this and other related NDDs. (NDDs) can arise from mutations in genes at each level of chromatin organization. Chromatin structure and
activity are regulated at a variety of levels. At the epigenomic level, writers, readers, and erasers of DNA
Therapeutic potential methylation and histone post-translational modifications covalently modify and read reversible DNA and
histone marks. Chromatin remodelers can slide or eject nucleosomes or exchange histone variants. Proteins
SETD1A (KMT2F) encodes one of two cata-
involved in chromatin organization provide higher-order organization in topologically associated domains, and
lytic subunits of the SET1A/B COMPASS some play key roles in key cell cycle states, including mitosis.

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methyltransferase complex. This complex potential for performing therapeutic screens *Correspondence:
[email protected] (S.P. Pașca).
is responsible for histone H3K4 di- and in this system.
https://doi.org/10.1016/j.tcb.2021.09.001
trimethylation and is associated with tran-
scriptional activation. Mutations in SETD1A Advances in next-generation sequencing © 2021 Elsevier Ltd. All rights reserved.
have been implicated in schizophrenia, and and chromatin mapping technologies
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