HMGA1 acts as an epigenetic gatekeeper of ASCL2 and Wnt signaling during colon tumorigenesis

Abstract

Mutated tumor cells undergo changes in chromatin accessibility and gene expression, resulting in aberrant proliferation and differentiation, although how this occurs is unclear. HMGA1 chromatin regulators are abundant in stem cells and oncogenic in diverse tissues; however, their role in colon tumorigenesis is only beginning to emerge. Here, we uncover a previously unknown epigenetic program whereby HMGA1 amplifies Wnt signaling during colon tumorigenesis driven by inflammatory microbiota and/or Adenomatous polyposis coli (Apc) inactivation. Mechanistically, HMGA1 “opens” chromatin to upregulate the stem cell regulator, Ascl2, and downstream Wnt effectors, promoting stem and Paneth-like cell states while depleting differentiated enterocytes. Loss of just one Hmga1 allele within colon epithelium restrains tumorigenesis and Wnt signaling driven by mutant Apc and inflammatory microbiota. However, HMGA1 deficiency has minimal effects in colon epithelium under homeostatic conditions. In human colon cancer cells, HMGA1 directly induces ASCL2 by recruiting activating histone marks. Silencing HMGA1 disrupts oncogenic properties, whereas reexpression of ASCL2 partially rescues these phenotypes. Further, HMGA1 and ASCL2 are coexpressed and upregulated in human colorectal cancer. Together, our results establish HMGA1 as an epigenetic gatekeeper of Wnt signals and cell state under conditions of APC inactivation, illuminating HMGA1 as a potential therapeutic target in colon cancer.

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Introduction
Exquisite control of transcriptional networks that regulate plasticity
and other stem cell properties allow for tissue specification during
embryogenesis and tissue maintenance after birth (1–9). Often
referred to as “stemness” networks, genes involved in self-renewal
and plasticity are largely silenced in differentiated tissues, although
a subset remain active in adult stem cells where they contribute to
tissue regeneration during homeostatic conditions or following inju-
ry and other stressors (3–6). By contrast, neoplastic cells frequent-
ly corrupt these same transcriptional networks to foster aberrant
growth and differentiation (1–7, 9, 10). Moreover, tumor progres-
sion is associated with increased expression of genes controlling
stem cell properties, which may foster the emergence of highly plas-
tic tumor cells capable of metastatic progression, immune evasion,
and therapy resistance (1–7, 9, 10). While mechanisms responsible
for cell state during tissue regeneration and tumorigenesis remain
incompletely understood, it is clear that chromatin reorganization
and epigenetic alterations contribute to plasticity, self renewal, and
other stem cell properties (4, 11).
As a highly regenerative tissue and frequent site for cancer, the
colon epithelium provides a unique paradigm to study plasticity
and cell state during tumorigenesis. Colon epithelium comprises
an intricately organized hierarchy of epithelial cells maintained by
proliferative stem cells that reside at the base of crypts (3, 4, 6–8).
Moreover, it is among the most regenerative tissues of the body,
renewing itself every 3–5 days to maintain nutrient absorption
essential for life and provide a protective barrier from gut pathogens
and toxins. Stem cells at the crypt base in colon and small intesti-
nal epithelium are marked by the serpentine coreceptor for Wnt
signals, leucine-rich, repeat-containing G-protein–coupled receptor
5 (LGR5) (7, 8). Recent studies in murine small intestine also iden-
tified distinct populations of upper crypt cells, marked by fibroblast
growth factor binding protein 1 (FGFBP1) or LGR4, that regener-
ate all lineages, including LGR5+ cells, the latter of which requires
the Wnt agonist, R-spondin (12–14). In both small intestine and
Mutated tumor cells undergo changes in chromatin accessibility and gene expression, resulting in aberrant proliferation and
differentiation, although how this occurs is unclear. HMGA1 chromatin regulators are abundant in stem cells and oncogenic
in diverse tissues; however, their role in colon tumorigenesis is only beginning to emerge. Here, we uncover a previously
unknown epigenetic program whereby HMGA1 amplifies Wnt signaling during colon tumorigenesis driven by inflammatory
microbiota and/or Adenomatous polyposis coli (Apc) inactivation. Mechanistically, HMGA1 “opens” chromatin to upregulate
the stem cell regulator, Ascl2, and downstream Wnt effectors, promoting stem and Paneth-like cell states while depleting
differentiated enterocytes. Loss of just one Hmga1 allele within colon epithelium restrains tumorigenesis and Wnt signaling
driven by mutant Apc and inflammatory microbiota. However, HMGA1 deficiency has minimal effects in colon epithelium
under homeostatic conditions. In human colon cancer cells, HMGA1 directly induces ASCL2 by recruiting activating histone
marks. Silencing HMGA1 disrupts oncogenic properties, whereas reexpression of ASCL2 partially rescues these phenotypes.
Further, HMGA1 and ASCL2 are coexpressed and upregulated in human colorectal cancer. Together, our results establish
HMGA1 as an epigenetic gatekeeper of Wnt signals and cell state under conditions of APC inactivation, illuminating HMGA1 as
a potential therapeutic target in colon cancer.
HMGA1 acts as an epigenetic gatekeeper of ASCL2 and
Wnt signaling during colon tumorigenesis
Li Z. Luo,1 Jung-Hyun Kim,1,2 Iliana Herrera,1 Shaoguang Wu,3 Xinqun Wu,3 Seong-Sik Park,2 Juyoung Cho,2 Leslie Cope,4
Lingling Xian,1 Bailey E. West,1,5 Julian Calderon-Espinosa,1,6 Joseph Kim,1 Zanshé Thompson,1 Isha Maloo,1,7 Tatianna Larman,8
Karen L. Reddy,9 Ying Feng,10 Eric R. Fearon,10 Cynthia L. Sears,3,11,12 and Linda Resar1,4,5,6,7,8,11
1Division of Hematology, Department of Medicine, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 2Research Institute, National Cancer Center, Goyang-si, Gyeonggido, Republic
of Korea. 3Division of Infectious Diseases, Department of Medicine, 4Sidney Kimmel Comprehensive Cancer Center, Division of Biostatistics, 5Pathobiology Graduate Program, Department of Pathology,
and 6Human Genetics Graduate Program, Department of Genetics and Molecular Medicine, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 7Biochemistry and Molecular
Biology Program, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA. 8Department of Pathology and 9Department of Biological Chemistry, the Johns Hopkins University School of
Medicine, Baltimore, Maryland, USA. 10Department of Oncology, University of Michigan, Ann Arbor, Michigan, USA. 11Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, and 12Molecular
Immunology, the Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Related Commentary: https://doi.org/10.1172/JCI187442
Conflict of interest: CS reports grant funding administered through her institution
from Janssen and Bristol Myers Squibb. She reports unrelated royalties from Up
to Date. LR reports investigator-initiated grant funding administered through her
institution from PharmaEssentia for an unrelated project.
Copyright: © 2025, Luo et al. This is an open access article published under the terms
of the Creative Commons Attribution 4.0 International License.
Submitted: July 11, 2024; Accepted: November 27, 2024; Published: February 3, 2025.
Reference information: J Clin Invest. 2025;135(3):e184442.
https://doi.org/10.1172/JCI184442.

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cell niche during epithelial regeneration (4, 30). However, HMGA1
function in colon stem and progenitor cells during tumorigenesis
was previously unknown.
Here, we uncover a previously unknown role for HMGA1 in
modulating transcriptional networks to expand LGR5+ stem cells
and Paneth-like cells during tumorigenesis driven by Apc deficiency.
Strikingly, loss of just a single Hmga1 allele disrupts tumorigenesis
while prolonging survival in two different models of colon tum-
origenesis with APC inactivation, including mice with biallelic
deletion of colonic epithelial Apc (CDX2P-CreERT2Apcf l/fl) (73, 74)
and mice harboring monoallelic mutant Apc (ApcMin/– or Min mice)
colonized with the inflammatory human symbiote, enterotoxigen-
ic Bacteroides fragilis (ETBF) (75–79). Single cell RNA sequencing
(scRNA-seq) in Apc-deficient crypt epithelium reveals that HMGA1
maintains colon crypt cells in a stem and Paneth-like cell state while
depleting differentiated enterocytes. Integration of transcriptomic
analyses with assays of chromatin accessibility demonstrate that
HMGA1 activates Wnt signals by “opening” chromatin at gene
loci governing Wnt signaling, including the stem cell regulator,
Achaete-Scute Family BHLH Transcription Factor 2 (Ascl2), in addition
to Wnt agonist receptors (Lgr5, Lrp5) and downstream effectors. We
focus on the gene encoding ASCL2 as a master regulator of cell fate
in the small intestine, although its role in the colon was previously
unknown. In human colon cancer cells, HMGA1 directly induces
ASCL2 by recruiting activating histone marks. Further, silencing
HMGA1 disrupts oncogenic properties (proliferation and clonoge-
nicity), while reexpression of ASCL2 partially rescues oncogenic
phenotypes in HMGA1-depleted human colon cancer cells. Most
importantly, both HMGA1 and ASCL2 are coexpressed and upregu-
lated in human colon cancer. Surprisingly, HMGA1 depletion has
minimal effects on colon epithelial regeneration under homeostatic
conditions. Our results establish HMGA1 as an epigenetic gate-
keeper of ASCL2 and Wnt signals in colon stem cells during tum-
origenesis, but not steady state homeostasis, highlighting HMGA1
pathways as promising therapeutic targets for colon carcinogenesis.
Results
Loss of just a single Hmga1 allele is sufficient to decrease tumorigenesis
and prolong survival in mice with colon tumors driven by biallelic Apc
inactivation. Because previous studies from our group and others
showed that HMGA1 is highly overexpressed in human colon can-
cer (30, 35, 62) and required for oncogenic properties in colon
cancer cell lines (35), we sought to assess its role in colon tumor-
igenesis in vivo. Since APC is the most commonly mutated gene
in human colon cancer (15), we examined CDX2P-CreERT2Apcfl/
fl mice, an established model of colon tumorigenesis caused by
inducible, biallelic loss of Apc within colon epithelium (73, 74).
CDX2P-CreERT2Apcfl/fl mice were crossed to mice with global Hmga1
deficiency (heterozygous or homozygous). Importantly, mice with
heterozygous Hmga1 have normal development and lifespans,
whereas those with homozygous deficiency have partial embry-
onic lethality and develop premature aging phenotypes (kyphosis,
bone loss, greying, and shortened lifespans) beginning after 10–12
months of age (29, 56). As expected, CDX2P-CreERT2Apcfl/fl mice
with Hmga1 heterozygous or homozygous deficiency have low-
er Hmga1 gene expression and protein levels in colon epithelium
(Supplemental Figure 1, A and B; supplemental material available
colon, a Wnt gradient maintains LGR5+ cells by repressing differ-
entiation at the base where Wnt levels are highest, while allowing
cells to differentiate as they move up the crypt with decreasing Wnt.
Thus, tightly regulated Wnt signaling is fundamental to epithelial
regeneration in the gut.
Not surprisingly, mutations that activate Wnt signals are com-
mon in colon adenomas and adenocarcinomas (15–22). Inactivat-
ing mutations in the gene encoding the Adenomatosis Polyposis
Coli (APC) tumor suppressor protein, first described in the familial
adenomatosis polyposis (FAP) syndrome, are the most common
genetic lesions found in colon adenomas and carcinomas (15–22).
APC normally restrains Wnt function by maintaining β-catenin in
the cytoplasm, thereby preventing β-catenin entry into the nucle-
us to activate Wnt target genes together with the TCF-4 transcrip-
tion factor. Genomic studies established a model whereby colon
carcinomas develop from polyps harboring APC mutations after
the stepwise accumulation of mutations that inactivate additional
tumor suppressor genes and/or activate protooncogenes (15–16).
Neoplastic polyps are thought to arise in colon stem cells express-
ing LGR5, although targeting mutated LGR5+ cells in therapy has
not been feasible (7, 8). Importantly, colon cancer is the third lead-
ing cause of cancer-related deaths in the US, and the incidence is
rising globally, particularly in younger individuals (23–26). Thus,
studies are warranted to decipher changes in cell state occurring
during colon tumorigenesis to identify mechanisms that could be
targeted to intercept the transition from mutant cells and localized
tumors to advanced disease.
High Mobility Group A (HMGA1) proteins are architectur-
al transcription factors that bind AT-rich sequences where they
modulate chromatin structure and gene expression (4, 27–55). The
HMGA1 gene is highly expressed during embryogenesis (4, 9, 34,
37) and in adult stem cells (4, 29, 30, 47), but is silenced in most
differentiated cells. HMGA1 becomes reexpressed in aggressive can-
cer cells and high levels portend adverse clinical outcomes (28–38,
40–43, 56–64). In colon cancer, HMGA1 is among the genes most
highly overexpressed compared with nonmalignant colon epithe-
lium (4, 30, 57, 62). While mechanisms upregulating HMGA1 in
cancer are incompletely understood, increasing evidence suggests
that diverse oncogenic pathways, including growth factors (65,
66), mutations — such as mutant Apc (67, 68), KRAS (28, 36, 69),
or mutant JAK2 (29) — and oncogenic transcription factors, like
cMYC (70) or cJUN (71, 72), converge on HMGA1 to induce its
expression in distinct settings. In transgenic mouse models, Hmga1
overexpression leads to tumorigenesis (35, 41, 42, 58, 64). For
example, transgenic mice overexpressing Hmga1 in lymphoid cells
develop clonal expansion with evolution to leukemia by upregu-
lating transcriptional networks active in proliferating stem cells,
poorly differentiated cancer cells, and inflammation (37, 38, 41, 58,
64). In experimental models of pancreatic cancer and myeloprolif-
erative neoplasms, HMGA1 activates gene networks in a cell-intrin-
sic fashion to drive aberrant proliferation and differentiation, while
inducing signals within the tumor microenvironment that promote
fibrosis, culminating in tumor progression (28, 29). HMGA1 also
upregulates genes involved in an epithelial-to-mesenchymal tran-
sition in colon cancer cell lines (35). In small intestinal stem cells,
HMGA1 induces Sox9 and Wnt signals from the stromal and epi-
thelial niches to maintain the stem cell compartment and Paneth

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Furthermore, ETBF colonization is common in colon cancer (up
to 90%) and epidemiologic studies suggest that it increases the risk
of carcinogenesis (75–85). Following inoculation with ETBF at
5–6 weeks of age, Min mice with intact Hmga1 exhibit poor weight
gain and robust distal colon tumorigenesis by 11–12 weeks with
a median survival of 17 weeks; by contrast, Min mice with glob-
al Hmga1 hemizygosity gain more weight, develop fewer tumors,
and exhibit prolonged survival (Figure 4, A–D). Histologic exam-
ination shows hyperproliferative colon epithelium and adeno-
matosis with increased crypt depth in the distal colons of Min
mice with intact HMGA1 compared with those with HMGA1
deficiency (Figure 4, E and F). As expected, HMGA1 mRNA and
protein levels by IHC are increased in mice with intact HMGA1
compared with Hmga1 haploinsufficient Min mice (Figure 4, E
and F, Figure 5, A–C, and Supplemental Figure 2A). Similarly,
the proportion of cells staining positive for intranuclear β-cat-
enin and cytoplasmic β-catenin are greater in colon epithelium
and tumors of ApcMin/+ mice with intact HMGA1, although Ki67
was unchanged in mice with intact or haploinsufficient HMGA1
(Figure 4, E and F, and Supplemental Figure 2B). Intriguingly,
HMGA1 protein staining is similar in tumors from ApcMin/+ mice
with intact HMGA1 and HMGA1 haploinsufficiency, suggesting
that mice with HMGA1 haploinsufficiency can upregulate the
intact Hmga1 allele to increase HMGA1 levels within their tumors
(Figure 5, A–C). The small intestinal tumor burden is also greater
in ApcMin/+ mice with intact HMGA1 (Supplemental Figure 2C),
indicating that HMGA1 contributes to tumorigenesis in both the
colon and small intestine of Min mice.
Loss of a single Hmga1 allele within the colon epithelium is sufficient
to reduce colon tumorigenesis induced by ETBF in Min mice. To deter-
mine whether HMGA1 deficiency within colon epithelium is suf-
ficient to mitigate tumorigenesis in the Min-ETBF model, we gen-
erated Min mice with Hmga1 deficiency (hetero- and homozygous
genetic deletion) restricted to colon and small intestinal epithelium
by crossing ApcMin/+ mice with Hmga1fl/fl mice on a Vil-cre back-
ground. Notably, Min mice with tissue-specific HMGA1 deficiency
developed fewer colon tumors and decreased crypt depth compared
with Min mice with intact HMGA1 (Figure 6, A–E). Surprisingly,
Min mice with Hmga1 haploinsufficiency had a similar decrease in
colon tumor number as Min mice with homozygous Hmga1 loss,
suggesting that a relatively modest decrease in HMGA1 within the
epithelial compartment alone is sufficient to mitigate tumorigene-
sis. Small intestinal tumors also decrease modestly in this model,
but only with homozygous loss of Hmga1 (Supplemental Figure
3A). By contrast, tissue-specific biallelic loss of Hmga1 in colon
crypts from WT mice lacking Apc mutation show no significant
changes in crypt depth, suggesting that HMGA1 deficiency under
steady state, homeostatic conditions (no ETBF colonization) is not
deleterious to colon epithelial regeneration (Supplemental Figure
3B). Together, these findings demonstrate that HMGA1 within the
crypt epithelium drives tumorigenesis, and, moreover, tissue-specif-
ic, Hmga1 haploinsufficiency is sufficient to impair colon tumor for-
mation driven by mutant Apc and inflammatory ETBF, highlighting
HMGA1 as a promising potential therapeutic target.
HMGA1 expands colon stem cells and Paneth-like cells while depleting
more differentiated enterocytes in Apc-deficient colon crypts. To investi-
gate molecular mechanisms underlying HMGA1 in Apc-deficient
online with this article; https://doi.org/10.1172/JCI184442DS1).
Following induction of Cre recombinase-mediated Apc deletion by
tamoxifen (TAM), CDX2P-CreERT2Apcfl/f l mice with intact Hmga1
alleles develop epithelial hyperplasia in the cecum, proximal, and
midcolon regions (Figure 1A) associated with weight loss by 4
weeks (Figure 1B) and decreased survival (median survival 43 days
after TAM; n = 16) (Figure 1C). Strikingly, loss of just a single
Hmga1 allele in CDX2P-CreERT2Apcfl/fl mice mitigates weight loss
while prolonging survival (median survival 61.5 days; P < 0.0001,
n = 12; Figure 1, A–C). Survival is prolonged further (median sur-
vival 78 days; P < 0.0001, n = 7) (Figure 1C) in mice with Hmga1
homozygous deficiency, indicating that Hmga1 gene dosage modu-
lates tumor progression in this model.
To determine more precisely how HMGA1 modulates tumori-
genesis in CDX2P-CreERT2Apcfl/fl mice, we compared colon weights
as a surrogate for tumor burden since extensive, contiguous tumors
in the proximal colon precludes precise enumeration. Both the
absolute and relative colon weight (% colon weight/body weight)
increase in CDX2P-CreERT2Apcfl/fl mice with intact Hmga1 compared
with those with heterozygous or homozygous Hmga1 deficiency
(Figure 1D and Supplemental Figure 1C). At 21 days following Apc
inactivation, colon epithelium becomes thickened and dysplastic
with extensive adenomatous changes and increased crypt depth in
CDX2P-CreERT2Apcfl/fl mice with intact Hmga1 (Figure 1, A and E,
and Figure 2, A and B). Intranuclear HMGA1 is prominent through-
out the crypts up to the luminal enterocytes in CDX2P-CreERT2Apcfl/f l
mice with intact Hmga1 by IHC. By contrast, intranuclear HMGA1
is normally restricted to the crypt bases in WT mice lacking Apc-defi-
ciency (Supplemental Figure 1D). Since APC restrains Wnt signals
by sequestering β-catenin within inhibitory, cytoplasmic complexes,
we compared β-catenin levels and localization in nuclei and cyto-
plasm in the Apc-deficient models. Following Apc inactivation (day
21), both nuclear and cytoplasmic β-catenin levels increase in colon
epithelial cells of mice with intact HMGA1 compared with those
with heterozygous or homozygous Hmga1 deficiency, paralleling
the distribution of intranuclear HMGA1 (Figure 2, A and B, and
Supplemental Figure 1E). Cells staining positive for the proliferation
marker Ki67 are also increased in CDX2P-CreERT2Apcfl/fl mice with
intact HMGA1 compared with those with HMGA1 deficiency early
in tumorigenesis, although they predominate at the crypt bases (Fig-
ure 2, A and B). In addition, HMGA1 increases in the adenomatous
epithelium compared with nontumor, midcolon epithelium in mice
with intact HMGA1 (Figure 3, A and B, and Supplemental Figure
1, A and B). Intriguingly, HMGA1 also increases in tumors from
mice with Apc inactivation and Hmga1 heterozygous deficiency com-
pared with adjacent nontumor colon epithelium (Figure 3, A and B,
and Supplemental Figure 1, A and B). These findings indicate that
loss of just a single Hmga1 allele in the setting to Apc inactivation
decreases hyperproliferation, β-catenin levels, and tumorigenesis.
HMGA1 hemizygous deficiency mitigates colon tumorigenesis induced
by ETBF in Min mice. Next, we investigated HMGA1 function in
the multiple intestinal neoplasia (Min) model, which harbors a
heterozygous Apc loss-of-function mutation (ApcMin/+ or Min+/–)
and develops distal colon tumors following inoculation with the
human symbiotic bacterium ETBF. This model recapitulates salient
features of human colon tumors with respect to the mutational
status, location in the distal colon, and histopathology (75–79).

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myeloid lineages (macrophage-like, mast cells, and neutrophils)
(Supplemental Figure 4A).
To dissect HMGA1-dependent changes in the cell of origin
for colon tumors, we focused on the epithelial island. With intact
HMGA1, the LGR5+ stem cell population, defined by high lev-
els of Lgr5, Msi1, Bmi1, and other stem cell transcripts (Supple-
mental Table 1), comprise the majority of cells (40.1%) within
CDX2P-CreERT2Apcfl/fl colon crypts (Figure 7D). Transit amplify-
ing (TA) cells are the next most abundant population, constitut-
ing 26% of epithelial crypt cells, whereas Paneth-like cells, based
on Paneth cell markers (Lyz1, Mmp7, Sox9, Retnlb, Chil3, Reg3g,
and Deta; Supplemental Table 1), comprise 20% of crypt cells.
Intriguingly, while Paneth cells are not present in normal colon
epithelium, Paneth cell “metaplasia” has been reported in proxi-
mal colon epithelium in adenomas, adenocarcinoma, and inflam-
matory bowel disease, and ectopic Paneth cells were observed
in colon epithelium of Apc-deficient mouse models (73, 74, 85,
86). The terminally differentiated enterocyte (EC) and goblet cell
colon crypts, we performed scRNA-seq in proximal colon crypt
cells from CDX2P-CreERT2Apcfl/fl mice with intact Hmga1 compared
with those with heterozygous or homozygous Hmga1 deficiency.
We examined transcriptomes at early stages in tumorigenesis (21
days following Apc inactivation via TAM) to identify mechanisms
involved in tumor initiation. Single-cell transcriptomes depicted by
uniform manifold approximation and projection (UMAP) reveal
differences in overall distribution in cells with or without HMGA1
(Figure 7A). Unsupervised hierarchical clustering of transcripts
(via Seurat) revealed 12 clusters (Figure 7B) from which cell iden-
tities were imputed using established markers (Supplemental Table
1). Of these clusters, five are comprised of epithelial crypt cells
(denoted epithelial island) based on expression of the colon epithe-
lial cell adhesion marker gene (Epcam), colon stem and progenitor
cell genes (Lgr5, Sox9, Ctnnb1), and proximity by UMAP (Figure
7C). The remaining clusters are comprised of immune cells (Figure
7B). Within the immune cell islands, we identified Cd4+ and Cd8+
T cells with smaller populations of B cells, macrophages, and other
Figure 1. Loss of a single Hmga1 allele mitigates colon tumorigenesis and prolongs survival in CDX2P-CreERT2/Apcfl/fl mice. (A) Representative
images (H&E) of proximal colon in CDX2P-CreERT2/Apcfl/fl mice with Hmga1 intact (Hmga1+/+ top), heterozygous deletion (Hmga1+/–, middle), or
homozygous deletion (Hmga1–/–, bottom) at survival endpoint necropsy (top: day 35 after TAM; middle: day 57; bottom: day 81). Scale bars: 250
μm. (B) Relative weight changes in CDX2P-CreERT2/Apcfl/fl models after TAM. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 1-way ANOVA).
(C) Kaplan-Meier plot showing survival in CDX2P-CreERT2/Apcfl/fl mice with Hmga1+/+, Hmga1+/–, or Hmga1+/+. (****P < 0.0001; Mantel-Cox test). (D)
Relative colon weight to body weight in CDX2P-CreERT2/Apcfl/fl mice with Hmga1+/+, Hmga1+/–, or Hmga1–/– (*P < 0.05; Hmga1+/+ versus Hmga1+/–,
**P < 0.01, Hmga1+/+ versus Hmga1–/–; Tukey’s multiple comparison test following significance by 1-way ANOVA). (E) Proximal colon crypt depth in
CDX2P-CreERT2/Apcfl/fl models (*P < 0.05, ***P < 0.01, ****P < 0.0001; 1-way ANOVA with Tukey’s multiple comparison test). Each shape (circle,
square, triangle) corresponds to a different mouse (n = 2–3/genotype). The solid shapes show the mean from each mouse; the open, smaller shapes
represent individual measurements/crypt (range = 9–13 crypts/mouse) at × 20 magnification.

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time = 0 to the most dedifferentiated, stem cell cluster (via Seurat,
Monocle 2). Intact HMGA1 results in a greater proportion of cells
at earlier stages in development (time = 0; undifferentiated stage)
whereas HMGA1 deficiency leads to more cells in later stages (time
= 12; more differentiated stage) within the differentiation trajectory
(Figure 8A). Next, we applied cell state analysis (Seurat, Monocle
2) as a more static assessment of differentiation status of each cell
along the trajectory in Apc-deficient epithelial crypt cells (Figure
8B). Cell states (defined by the top 200 most differentially expressed
genes within 5 groups with distinct transcriptomes) were assigned
to individual cells along the trajectory. Similar to our cluster anal-
ysis, Apc-deficient crypt cells with intact Hmga1 include a greater
proportion of cells in an undifferentiated stem cell state (state 0)
or Paneth-like state (state 1) compared with those with HMGA1
deficiency, which skews development to later, more differentiated
cell states (states 3–4) (Figure 8B) or ECs (Figure 8C).
Single cell transcriptomes suggest that HMGA1 accelerates prolifera-
tion by inducing gene networks involved in cell cycle progression. Next,
we inferred cell cycle status of each cell in the epithelial cluster
from scRNA-seq (Seurat; standard settings). Transcriptomic chang-
es suggest that intact HMGA1 in Apc-deficient crypt epithelial cells
function by increasing proliferation, as evidenced by decreases
in the proportion of cells in G0/G1 concurrent with increases in
the proportion reaching G2/M; the proportion of S phase cells
were similar in Apc-deficient crypt cells with or without HMGA1
clusters comprise the least frequent crypt cell types in this mod-
el (9.7% and 4.1%, respectively). Strikingly, HMGA1 deficiency
decreases the proportion of stem and Paneth-like cell clusters by
about 50% (P < 0.0001) together with a concurrent expansion in
the proportion of differentiated ECs (from 9.7% to 28.9%; P <
0.0001), TA cells (from 26% to 34.9%; P < 0.0001), and goblet
cells (from 4.1% to 8%; P < 0.0001) within the crypt epithelium.
In Apc-deficient crypts with Hmga1 heterozygous deficiency, the
changes in most clusters are intermediate between crypts with
intact or homozygous deficiency of Hmga1 (Supplemental Figure
4, B and C). Together, these results indicate that intact HMGA1
is required to maintain the colon stem and Paneth-like cells in the
setting of Apc deficiency while depleting more differentiated cells
(ECs and goblet cells).
Within the immune cell islands, Cd4+ and Cd8+ T cells increase
in frequency in the HMGA1-deficient crypt cells (Supplemental
Figure 4), the latter of which could reflect an increase in tumor-in-
filtrating T lymphocytes. Both Cd4+ and Cd8+ T cells with Hmga1
genetic deletion also exhibit a shift on UMAP, indicating that
HMGA1 loss within these T cell populations alters their underlying
transcriptomes (Supplemental Figure 4A).
Trajectory and cell state analyses show that HMGA1 maintains an
earlier cell state in Apc-deficient crypt cells. To delineate HMGA1 func-
tion in differentiation dynamics in Apc-deficient colon crypt epi-
thelium, we performed pseudotime trajectory analyses, assigning
Figure 2. Hmga1 haploinsufficiency decreases β-catenin and Ki67 in CDX2P-CreERT2/Apcfl/fl mice. (A) Representative IHC images of nuclear HMGA1 (top),
β-catenin (middle), and Ki67 (bottom) in CDX2P-CreERT2/Apcfl/fl models at 3 weeks after TAM. Scale bar: 200 μm. (B) Quantitative comparisons of IHC
images (*P < 0.05, **P < 0.01; Tukey’s multiple comparison test following significance by 1-way ANOVA). Each shape (circle, square, triangle) corresponds
to a different mouse [top bar graph (n = 2–3/genotype), middle bar graph (n = 3/genotype), bottom bar graph (n = 3/genotype)]. The solid shapes show the
mean from each mouse; the open, smaller shapes represent individual values/field (range = 8–24 fields/mouse) at × 20 magnification.

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deficiency (Supplemental Figure 5). The proliferation marker gene
encoding Ki67 is among the most upregulated genes of all G2/M
genes. Collectively, our single-cell transcriptomes, together with
increases in crypt depth, Ki67 protein staining, and tumorigenesis
in mice with colon epithelial Apc inactivation and intact HMGA1
(Figure 1) are consistent with a model whereby HMGA1 increases
proliferation restricted to cells at the earliest developmental stages,
leading to expansion in stem and Paneth-like cells at the expense of
more differentiated cells within the crypt epithelium.
HMGA1 activates gene networks within crypt epithelial cells involved
in IFN signaling, inflammation, DNA repair, proliferation, and Wnt
signaling. To elucidate mechanisms underlying HMGA1 in Apc-
deficient crypt cells, we performed gene set enrichment analysis
(GSEA; MSigDB) with Hallmark and Curated gene sets (87, 88).
GSEA with transcripts from all clusters (epithelial and immune cells)
reveal that HMGA1 upregulates heterogenous pathways, including
those associated with metabolism (oxidative phosphorylation and
glycolysis), proliferation (MYC Targets V1 and MYC Targets V2),
and inflammation (IFN-α Response and IFN-γ Response) (Supple-
mental Figure 6). HMGA1 also activates multiple WNT networks,
including Wnt Pathway requiring MYC, Degradation of the β-cat-
enin Destruction Complex, TCF Dependent Signaling in Response
to WNT, and APC Targets (Table 1). By contrast, transcriptional
networks repressed by HMGA1 include allograft rejection, IL2-
STAT5 signaling, and mitotic spindle genes (Hallmark). Notably,
repression in allograft rejection and IL2-STAT5 gene networks
have been implicated in immune escape and decreases in cytotoxic
tumor infiltrating lymphocytes (Supplemental Figure 6) (89). The
heterogeneity in these pathways is consistent with the diverse cell
populations (epithelial and immune) within the crypts.
To focus our analysis on the tumor-initiating cells, we per-
formed GSEA exclusively on transcripts from the crypt epithe-
lial island. Further, this island is comprised of the majority of
cells from the crypt isolates and cell numbers are sufficient for
pathway analyses. In the remaining immune islands, cell numbers
were insufficient for further GSEA. Strikingly, HMGA1 activates
transcriptional networks involved in inflammation, including
IFN-α and IFN-γ response genes and proliferation (MYC targets
V1) within the epithelial island (Figure 9A). DNA repair genes
are also induced, which is a frequent transcriptional response
when quiescent stem cells are triggered to cycle and proliferate
(Figure 9A) (90–92). Among the IFN networks, multiple IFN-in-
duced genes that mediate inflammatory signals are upregulated
by HMGA1, including IFN-induced transmembrane proteins 1, 2, 3
(Ifitm 1–3), IFN stimulated gene 15 (Isg15), Stat1, Stat2, and cyto-
kines (Ccl5, Cxcl9/10) (Figure 9B). Wnt pathway genes are also
prominent among the networks activated by HMGA1 within the
epithelial crypt cells (Figure 9A and Table 1). By contrast, HMGA1
represses gene networks involved in fatty acid metabolism and
adipogenesis, metabolic pathways used extensively by differen-
tiated ECs in intestinal epithelium (Figure 9A) (93). HMGA1
also represses genes controlling protein secretion (Figure 9A), an
important cellular function of differentiated ECs, which secrete
digestive enzymes (93). Together, our single-cell transcriptomes
Figure 3. Hmga1 deficiency decreases colon tumorigenesis. (A) Representative IHC images of nuclear HMGA1 (top), β-catenin (middle), and Ki67 (bottom) in
CDX2P-CreERT2/Apcfl/fl models at 3 weeks after TAM. Scale bars: 1,000 μm (top panel); 200 μm (lower panel). (B) Quantitative comparisons of IHC images (*P
< 0.05, ***P < 0.001, ****P < 0.0001; Tukey’s multiple comparison test following significance by 1-way ANOVA). Each shape (circle, square, triangle, hexagon)
corresponds to a different mouse (n = 3–4/genotype). The solid shapes show the mean values from each tumor; the open, smaller shapes represent individual
values/field (range = 6–17 fields/tumor from 1–5 tumors/mouse) at × 20 magnification.

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and pathway analysis demonstrate that HMGA1 expands the
stem and Paneth-like cells while driving transcriptional networks
involved in proliferation, inflammation, Wnt signaling, and DNA
repair. Conversely, HMGA1 restrains differentiation and represses
metabolic gene networks active in differentiated ECs.
HMGA1 enhances chromatin accessibility at gene loci involved in
proliferation, DNA repair, inflammation, and Wnt signaling. Because
HMGA1 is an architectural transcription factor that modulates
chromatin structure, we performed assays to detect accessible chro-
matin mediated by HMGA1 via assays of transposase-accessible
chromatin sequencing (ATAC-seq) in Apc-deficient crypt cells with
or without HMGA1 deficiency (94). Notably, overall chromatin
accessibility is enhanced in Apc-deficient crypt cells with intact
HMGA1 compared with crypts lacking HMGA1 (Figure 10,
A–C). HMGA1 results in both more peaks and longer stretches of
accessible chromatin overall (Figure 10, A–C). Focusing on pro-
moter regions (up to –3 kb from the transcription start sites), we
also identified more peaks and longer stretches of open chromatin
within these regulatory regions with intact HMGA1 (Figure 10, B
and C). Similar to our scRNA-seq results, gene networks associated
Figure 4. Hmga1 haploinsufficiency disrupts colon tumorigenesis induced by ETBF in APCMin/+ mice. (A) Body weights at necropsy after ETBF in ApcMin/+
mice with intact Hmga1 or heterozygous Hmga1 (*P < 0.05, **P < 0.01, ***P < 0.001; student’s t test). (B) Representative images of methylene-blue
stained colons to visualize tumors in ApcMin/+/Hmga1+/+ mouse (top) compared with ApcMin/+/Hmga1+/– mouse (bottom) at 11–12 weeks after ETBF. (C) Nor-
malized tumor numbers in ApcMin/+ models (*P < 0.05; Mann-Whitney test). (D) Kaplan-Meier plot showing survival in in ApcMin/+ mice with intact Hmga1
or heterozygous (*P < 0.05; Mantel-Cox test). (E) Representative images (H&E left; IHC right; Scale bars: 200 μm) for HMGA1 (second column), β-catenin
(third column), and Ki67 (right) in distal colon of ApcMin/+ models at 11–12 weeks after ETBF. (F) Comparison of crypt depths (**P < 0.01) and IHC for nuclear
HMGA1 (**P < 0.01), nuclear β-catenin (****P < 0.0001) and Ki-67(P = 0.16, unpaired student’s t test for each comparison) in ApcMin/+ models. For crypt
depth (left), each shape (circle, square, triangle, hexagon) corresponds to a different mouse (n = 3–4/genotype). The solid shapes show the mean from
each mouse; the open, smaller shapes represent individual measurements/crypt (range = 9–16 crypts/mouse). For the IHC comparisons, each shape (circle,
square, triangle, hexagon) corresponds to a different mouse (n = 3–4/genotype), the solid shapes show the mean value from each mouse; the open, small-
er shapes represent individual values/field (range=9-19 fields/mouse) at x20 magnification.

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Tcf4, Prom1, Lgr5, and Lrp5 (Supplemental Figure 8). Intriguingly,
HMGA1 is also associated with accessible chromatin at the Hmga1
promoter (Supplemental Figure 8), suggesting that high levels
of HMGA1 induce its own expression in Apc-deficient crypts by
opening chromatin at its promoter region. Together, these results
demonstrate that HMGA1 enhances chromatin accessibility to
activate Wnt agonist receptor signaling and Wnt effector genes. In
small intestinal epithelium, ASCL2 activates Wnt genes (95–98),
and upregulation in Ascl2 could trigger a feed-forward loop whereby
HMGA1 activates Ascl2, which, in turn, amplifies Wnt gene expres-
sion in colon epithelium with Apc inactivation.
HMGA1 and ASCL2 are upregulated and coexpressed in human col-
orectal cancer. To determine which HMGA1 pathways are relevant
to human colon tumorigenesis, we queried the Cancer Genome
Atlas (TCGA) for expression of HMGA1 and Wnt genes (Fig-
ure 11, C and D). As we previously reported, HMGA1 and SOX9
are upregulated in colon cancer compared with nonmalignant
epithelium (4, 30, 62). Strikingly, most of the Wnt genes upreg-
ulated by HMGA1 in our murine model are also upregulated in
human colon cancer, including the WNT effectors, ASCL2, AXIN2,
CTNNB1, MYC, EPHB2, CD44, and ETS2 and the WNT receptors,
LGR5, LRP5, and LRP6 (Figure 11, C and D). Further, both ASCL2
and cMYC are upregulated and positively correlated with HMGA1,
suggesting that HMGA1 may directly induce their expression in
human colon tumorigenesis (Figure 11C).
HMGA1 upregulates ASCL2 and promotes oncogenic properties
in human colon cancer cells. The ASCL2 transcription factor is
critical to cell fate in the small intestine (95–98) although its
with HMGA1-mediated accessible chromatin included pathways
involved in proliferation (MYC Targets V1, E2F Targets, G2M
Checkpoint genes) and inflammation (IFN-γ, TNF-α signaling via
NF-κB (Figure 10, D and E, and Supplemental Figure 7). Acces-
sible chromatin is also enriched at Wnt signaling gene networks
(Table 1). Intersecting pathways identified by both ATAC-seq and
scRNA-seq (epithelial island) revealed that HMGA1 increased
chromatin accessibility and expression of genes involved in pro-
liferation (MYC Targets V1), DNA repair, inflammation (IFN-γ
response genes), and Wnt signaling (Figure 10E and Table 1).
HMGA1 amplifies expression of Wnt pathway genes in Apc-de-
ficient colon crypts. Given the fundamental role for Wnt signaling
in colon tumorigenesis and HMGA1-dependent upregulation of
Wnt genes and β-catenin levels in our tumor models, we further
examined the relationship between HMGA1 and Wnt genes. We
focused on canonical Wnt pathway genes, including Wnt effectors
(Ctnnb1, Tcf4, Axin2, Cd44, Ets2, Ephb2, Ascl2, cMyc, Prom1, Sox9)
and Wnt receptors (Lgr5, Lrp5, Lrp6, Fzd5, Fzd7). Remarkably, all
Wnt effector genes were upregulated at the level of single cells in
the setting of intact HMGA1 and Apc deficiency (Figure 11A). Of
the Wnt receptors, both Lgr5 and Lrp5 transcripts are upregulat-
ed in crypt cells with intact HMGA1. We also found significant
positive correlations between Hmga1 and multiple Wnt effectors
(Figure 11B) with the strongest correlations (r > 0.68; P < 0.05) for
Ascl2, Axin2, Tcf4, Ctnnb1, Ephb2, and cMyc. To determine whether
HMGA1 enhances chromatin accessibility at promoter regions for
these genes, we examined our ATAC-seq results, which revealed
increased chromatin accessibility at promoter regions for Ascl2,
Figure 5. Hmga1 haploinsufficiency decreases β-catenin and tumorigen-
esis induced by ETBF in APCMin/+ mice. (A) Representative images (H&E)
of distal colon tumors in ApcMin/+ models at 11–12 weeks after ETBF. (B)
Representative images (IHC) of distal tumors in ApcMin/+ models for HMGA1
and β-catenin at 11–12 weeks after ETBF. Scale bars: 200 μm. (C) Quanti-
tative IHC comparisons of distal tumors in ApcMin/+ models for HMGA1 (P =
0.09) and β-catenin (****P < 0.0001; unpaired student’s t test for both).
Each shape (circle, square, triangle, hexagon) corresponds to a different
mouse (n = 3–4/genotype). The solid shapes show the mean from each
mouse; the open, smaller shapes represent individual values/field (range =
2–4 tumor fields/mouse) at × 20 magnification.

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or CRISPR/Cas9 represses ASCL2, demonstrating that ASCL2
expression depends on HMGA1 (Figure 12A and Supplemental
Figure 9A). Next, we tested whether HMGA1 deficiency affects
oncogenic properties in these cells. We previously reported that
HMGA1 knockdown decreases clonogenicity in SW480 cells
using plasmid-mediated gene silencing (35). Here, we found that
role in the colon has not been studied in detail. Because our
results strongly link HMGA1 to ASCL2 in colon tumorigenesis
in humans and mice, we tested whether HMGA1 directly acti-
vates ASCL2 expression in human colon cancer cells. Silencing
HMGA1 in 2 human colon cancer cell lines (SW620, SW480)
by lentiviral-mediated delivery of short hairpin RNA (shRNA)
Figure 6. Loss of Hmga1 allele within colon epithelium decreases colon tumorigenesis induced by ETBF in ApcMin/+ mice. (A) Representative images
of methylene-blue–stained colons of ApcMin/+ mice (top) compared with ApcMin with tissue-specific heterozygous Hmga1 deletion (middle) and
tissue-specific homozygous Hmga1 deletion (bottom) at 11–12 weeks after ETBF. (B) Relative tumor numbers (%) in ApcMin mice with intact Hmga1,
tissue-specific heterozygous Hmga1 deletion, or tissue-specific homozygous Hmga1 deletion from 3 separate experiments; tumor numbers in control
were assigned a value of 100 (*P < 0.05; Mann-Whitney test for both comparisons). (C) Representative images (H&E) of distal colon of ApcMin/+ with
or without tissue-specific Hmga1 deficiency models. Scale bars: 100 μm. (D) Distal colon crypt depths in ApcMin/+ mice with or without tissue-specific
Hmga1 deficiency. (**P < 0.01, ****P < 0.0001; Tukey ’s multiple comparisons test following significance by 1-way ANOVA). Each shape (circle, square,
triangle, hexagon) corresponds to a different mouse (n = 3–4/genotype). The solid shapes show the mean value from each mouse; the open, smaller
shapes represent individual measurements/crypt (range = 9–19 crypts/mouse) at × 20 magnification. (E) Representative images (H&E) of distal colon
tumors of ApcMin/+ with or without tissue-specific Hmga1 deficiency.

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cancer cell lines (SW620 and SW480) with HMGA1 silencing
(Supplemental Figure 9C). Restoration in ASCL2 levels results in
a partial rescue of proliferation and full rescue of clonogenicity
in both cell lines, suggesting that ASCL2 mediates some, but not
all, effects of HMGA1 in these colon cancer cells (Supplemental
Figure 9, C and D).
HMGA1 silencing (via shRNA or CRISPR) disrupts proliferation
and clonogenicity similarly in SW620 and SW480 cells (Figure
12, A and B, and Supplemental Figure 9, A and B), demonstrat-
ing that these in vitro, oncogenic phenotypes depend on high
levels of HMGA1. To determine whether ASCL2 restoration will
rescue these phenotypes, we reexpressed ASCL2 in both colon
Figure 7. HMGA1 expands colon stem cells and Paneth-like cells while depleting more differentiated cells in Apc-deficient colon crypts. (A) UMAPs from
scRNA-seq of crypt cells from CDX2P-CreERT2 Apcfl/fl mice with Hmga1+/+ or Hmga1–/–; shown together (left) or separately to highlight differences (center
and right). (B) UMAP from scRNA-seq by cluster. Three distinct islands capture epithelial cell types (red circle), T cells (blue circle), and other immune cells
(yellow). Imputed cell identities are designated by separate colors. TA, transit amplifying cells; EC, enterocytes. (C) Epcam, Lgr5, and other Wnt genes
(Sox9, Ctnnb1) are enriched in the epithelial island. Single cell transcripts from both genotypes are shown. (D) Relative proportion of cell types in crypt cells
by genotype (bar graph, left; Table, right). (Association between cell and HMGA1 status was evaluated by χ2 test for each cell type versus all others).

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tal Figure 10A). Because sites 6 and 7 are within 10 base pairs of each
other, they could not be resolved by ChIP-PCR, and were therefore
denoted region 6–7. Intriguingly, all of these sites are positioned near
the homologous regions of HMGA1-dependent accessible chroma-
tin in the mouse Ascl2 promoter (Figure 12C). By chromatin immu-
noprecipitation-PCR (ChIP-PCR), we assessed HMGA1 chromatin
HMGA1 directly induces ASCL2 by binding to its promoter and recruit-
ing activating histone marks in human colon cancer cells. To ascertain
whether HMGA1 binds directly to the ASCL2 promoter to activate
its expression, we used an in silico prediction algorithm (TRAP) (99),
which identified 7 potential HMGA1 binding sites within the ASCL2
promoter-enhancer region (labeled 1, 2, 3, 4, 5, 6, and 7; Supplemen-
Figure 8. Hmga1 deficiency alters cell state, decreasing stem and Paneth-like cell populations while expanding more differentiated cell populations
in Apc deficient crypt cells. (A) Pseudotime trajectory analysis estimated from scRNA-seq of CDX2P-CreERT2 Apcfl/fl crypt cells from the epithelial island
with Hmga1+/+ or Hmga1–/–. HMGA1 deficient cells are more prominent in later stages of pseudotime (indicated by black ovals) compared with time 0 cells.
(B) Cell states defined by the top 200 most differentially expressed genes on the trajectories from pseudotime analysis were assigned 0–4 and indicat-
ed by color on a trajectory plot (left) or bar graph (right). Note the skewing to cell states 3 and 4 in HMGA1 deficient cells. (C) Stem cells and enterocytes
(ECs) imputed from scRNA-seq are shown on the trajectories to highlight the major differences between CDX2P-CreERT2 Apcfl/fl cells with intact HMGA1 or
HMGA1 deficiency. HMGA1 deficient cells have increased ECs (blue) with decreased stem cells (violet). Bar graphs show relative cell frequencies (right); the
top graphs show only stem and ECs, the bottom includes all cells with grey depicting cells that are not stem cells nor ECs.

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the ASCL2 promoter, with greatest enrichment near the HMGA1
binding site number 1 (located near the transcription start site [TSS])
and these active marks are depleted with HMGA1 silencing (Fig-
ure 12D and Supplemental Figure 10C). By contrast, there was no
change with HMGA1 silencing in the positive control, histone H3,
which is a ubiquitous histone that does not modulate gene expres-
sion (Figure 12E and Supplemental Figure 10C). We also tested
whether HMGA1 depletion enables repressive histones to bind to
the ASCL2 promoter as a mechanism of downregulating ASCL2 with
HMGA1 silencing. Because the repressive histone 3 lysine 27 trimeth-
yl (H3K27me3) was identified in a colon cancer cell line (HCT116)
from a public database (GSE171817), we assessed its binding rela-
tive to that of HMGA1. In control SW620 cells with high HMGA1,
there was minimal binding of the repressive mark, H3K27me3; how-
ever, HMGA1 depletion results in modest, yet significant increases
in the H3K27me3 repressive mark at the ASCL2 promoter enhancer
region (Figure 12E and Supplemental Figure 10D).
To determine if HMGA1 activates the ASCL2 promoter, we
cloned the human ASCL2 promoter sequence (–2.5 kb from the
TSS) upstream of the luciferase reporter gene and transfected
occupancy at these sites in SW620 cells, as these cells have higher lev-
els of both HMGA1 and ASCL2 compared with SW480 cells. There
was robust HMGA1 occupancy throughout the ASCL2 promoter
compared with the IgG antibody as a negative control (Supplemen-
tal Figure 10, A and B). Next, we compared HMGA1 chromatin
binding in SW620 cells with or without HMGA1 silencing, which
showed enrichment for HMGA1 occupancy at the same regions
in control cells and depletion of HMGA1 with HMGA1 silencing,
validating the specificity of our HMGA1 antibody (Figure 12D and
Supplemental Figure 10C). Because HMGA1 recruits active his-
tone marks to upregulate developmental genes in other settings (28,
29), we tested whether HMGA1 binding associates with activating
histones, including histone H3 lysine 4 trimethylation (H3K4me3)
and histone H3 lysine 27 acetylation (H3K27Ac), which mark active
promoters and enhancers, respectively. These marks also associate
with HMGA1 chromatin binding in other tumor settings (28, 29).
We found enrichment for both activating histone marks (H3K4me3,
H3K27Ac) in the regions of HMGA1 binding in SW620 and SW480
cells from public databases (GSE10692) (Figure 12, C and D). By
ChIP-PCR, we found that both H3K4me3 and H3K27Ac bind to
Figure 9. HMGA1 activates gene networks within the crypt epithelial
island involved in IFN signaling, inflammation, DNA repair, prolifera-
tion, and Wnt signaling. (A) GSEA analysis (left) of single cell transcripts
from the epithelial island reveals that HMGA1 activates pathways
involved in inflammation (IFN-α, IFN-γ), DNA repair, and proliferation
(MYC) while repressing pathways active in differentiated ECs (fatty acid
metabolism, protein secretion); FDR ≤ 0.25. Enrichment plots (right) show
HMGA1 networks in more detail, including genes involved in inflammation
(IFN-α), DNA repair, and Wnt signaling. Normalized enrichment score
(NES) and normalized P values are indicated. (B) IFN-inducible genes that
mediate inflammatory signals, including IFN-induced transmembrane 1,
2, 3, (Ifitm1, 2, 3) genes, IFN stimulated gene 15 (Isg15), Stat1, Stat2, and
cytokines (Ccl5, Cxcl9, Cxcl10) are activated by HMGA1. Dot plots depict
gene expression (–0.4 to +0.4) and the proportion of cells (25%–75%)
expressing each transcript within the epithelial island.

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within the crypts along with evidence implicating mutated LGR5+
colon stem cells as a tumor initiating cell (100). Moreover, the inci-
dence of colon cancer is increasing globally, particularly in younger
individuals, highlighting the significance of this work (24–26).
Here, we discover that HMGA1 acts as an epigenetic regula-
tor that imposes a stem-like chromatin state within Apc-deficient
crypt epithelial cells. HMGA1 enhances chromatin accessibility
at key loci, leading to activation of gene networks involved in Wnt
signaling, proliferation, and inflammation early in tumorigenesis.
As an architectural transcription factor, HMGA1 binds to DNA
and recruits histones and other chromatin complexes to modulate
gene expression, rather than acting on its own. While HMGA1
drives clonal expansion, aberrant differentiation, and transforma-
tion in diverse settings, its function in colon tumorigenesis has
not been studied in detail despite the fact that it is among the
most overexpressed genes in colon cancer compared with nonma-
lignant epithelium (30, 62). We found that Hmga1 haploinsuffi-
ciency dampens colon tumor development and prolongs survival
in 2 models. Importantly, mice with Hmga1 heterozygosity (and
WT Apc) have normal development and lifespans (28, 29, 56).
By contrast, Apc deletion together with intact HMGA1 results in
increasing HMGA1 and nuclear β-catenin protein levels, not only
at their normal location at the crypt base, but throughout the crypt
extending toward the luminal epithelium. Intriguingly, HMGA1
protein levels increase within the colon tumors compared with
nontumor colon epithelium, even in the setting of Hmga1 hap-
loinsufficiency. Precisely how this occurs will require further
investigation, although these results underscore the importance
of HMGA1 in tumorigenesis in the CDX2P-CreERT2Apcfl/fl. While
prior studies show that HMGA2 is overexpressed in colon cancer
and Hmga2 drives tumorigenesis in mouse models with Let-7 defi-
ciency (101), we focus on HMGA1 since transcripts are approx-
imately 100-fold higher than HMGA2 in colon cancer datasets
(TCGA) and in many other human tumors (28–30).
In the Min model following inoculation with ETBF, Hmga1
haploinsufficiency globally or within the colon epithelium is suf-
ficient to decrease tumorigenesis. Surprisingly, complete loss of
Hmga1 (homozygous deficiency) from colon epithelium decreas-
es tumor incidence similar to that of haploinsufficiency. This
was unexpected, since global deletion of Hmga1 in the biallelic
this construct into colon cancer cell lines (SW620, SW480) (Sup-
plemental Figure 11A). In both cell lines with abundant levels of
HMGA1, the ASCL2 promoter is induced compared with control
vector lacking the ASCL2 promoter sequence (Supplemental Fig-
ure 11B). By contrast, HMGA1 silencing decreases ASCL2 promot-
er activity, consistent with HMGA1-dependent activation of the
ASCL2 promoter (Supplemental Figure 11B). Together, our results
support a model whereby HMGA1, present in high levels, binds to
the ASCL2 promoter, enhances chromatin accessibility, and recruits
activating histones to induce ASCL2 and downstream Wnt genes,
thereby driving tumorigenesis in the setting of Apc deficiency.
Moreover, our findings further highlight HMGA1 as a promising
potential therapeutic target, particularly since loss of HMGA1 in
colon epithelium has only subtle effects on epithelial regeneration
under homeostatic conditions.
Discussion
Changes in nuclear structure and function are required for normal
development, tissue regeneration, and tumorigenesis (4, 11). While
underlying mechanisms remain incompletely understood, chroma-
tin state has emerged as a fundamental player required for diverse
cell fate decisions in tumor biology. Embryonic stem cells and tis-
sue-specific, adult stem cells have large nuclei harboring “open”
accessible chromatin, which is thought to endow these cells with
developmental potency or the capacity to differentiate into diverse
progeny with distinct functions (4, 11). Similarly, nuclei in aggres-
sive cancer cells are often enlarged and irregular (4, 11), whereas
nuclear compaction accompanies differentiation in normal tissues
(4, 11). Though somatic mutations accumulate in adult stem cells
over time, particularly in highly proliferative tissues, such as the
colon crypts, most mutated cells do not evolve into tumors. Thus,
changes in chromatin structure and cell state provide a plausible
requisite for tumor development. Indeed, pathologists distinguish
cancer cells from nonmalignant cells primarily by alterations in
nuclear architecture. These observations suggest that understanding
mechanisms underlying chromatin structure and cell state during
tumor evolution could reveal strategies to intercept the transition
from early neoplasia to invasive cancers. Colon tumorigenesis offers
a unique opportunity to study cell state, adult stem cells, and tumor-
igenesis given the hierarchical organization of stem and progenitors
Table 1. Curated WNT pathways upregulated by HMGA1
Pathway
scRNA-seq (all clusters) scRNA-seq (epithelial clusters) ATAC-seq
Size NES
NOM
P value
FDR
q value Size NES
NOM
P value
FDR
q value Size NES
NOM
P value
FDR
q value
Sansom APC Targets Up 129 2.055 0.000 0.002 129 2.055 0.000 0.002 42 1.676 0.020 0.102
Sansom APC Targets Require MYC 220 1.792 0.000 0.043 220 1.463 0.000 0.200 49 1.751 0.000 0.089
Sansom WNT Pathway Require MYC 56 1.707 0.008 0.059 56 1.493 0.024 0.187 24 –0.663 0.836 1.000
Reactome Degradation of β-catenin by the
Destruction Complex 78 1.605 0.000 0.097 78 1.383 0.076 0. 249 17 1.913 0.017 0.060
Reactome β-catenin Independent WNT Signaling 110 1.421 0.030 0.187 110 1.219 0.097 0.358 22 1.326 0.109 0.265
Reactome TCF Dependent Signaling in Response
to WNT 136 1.395 0.021 0.207 136 1.033 0.319 0.532 36 0.917 0.553 0.700
Sansom APC Targets 196 1.390 0.005 0.210 196 1.002 0.390 0.581 70 1.371 0.100 0.246
Size, gene number per network; NES, normalized enrichment score; NOM P-value, normalized P-value.

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Figure 10. HMGA1 enhances chromatin accessibility at gene loci involved in proliferation, DNA repair, and inflammation. (A) HMGA1 increases chromatin
accessibility in crypt cell nuclei globally in CDX2P-CreERT2 Apcfl/fl mice. (B) HMGA1 enhances chromatin accessibility in promoter regions ranging from 0 to
–3 kb upstream of the transcription start sites shown by average peak lengths. (****P < 0.0001; student’s t test). (C) HMGA1 enhances chromatin accessi-
bility in promoter regions ranging from 0 to –3 kb upstream of the transcription start sites shown by number of significantly expanded peaks. (P < 0.0001;
χ2). (D) HMGA1 enhances chromatin accessibility in gene sets involved in proliferation, inflammation, and metabolism. (E) GSEA pathways identified by
intersecting ATAC-seq and scRNA-seq pathways with associated P values.

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Figure 11. HMGA1 amplifies Wnt genes in Apc-deficient colon crypts, and these HMGA1-Wnt pathways are activated in human colon cancer. (A) Dot
plot of Wnt effector and receptor gene expression in crypt cells of CDX2P-CreERT2 Apcfl/fl mice with Hmga1+/+ versus Hmga1–/–, demonstrating that HMGA1
activates all Wnt effectors and many Wnt receptor genes. (B) Hmga1 is positively and strongly correlated with Wnt genes, including Wnt effector genes
(Ascl2, Axin2, Tcf4, Ctnnb1, Myc, Ephb2, Sox9) and Wnt receptor genes (Lgr5, Lrp5, Fzd7) (Spearman’s rank correlation test). (C) Heatmap showing HMGA1
and WNT genes. HMGA1 correlates positively with ASCL2 and MYC (log scale) in human colon cancer. (D) HMGA1 and WNT genes in nonmalignant colon
epithelium (n = 41) and human colorectal adenocarcinoma (n = 286) from TCGA (student’s t test). †HMGA1 and SOC9 expression were previously reported in
ref. 30. TPM, transcripts per million. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Figure 12. HMGA1 induces ASCL2 by directly binding to the promoter and recruiting activating histone marks. (A) Silencing HMGA1 represses
ASCL2 and decreases proliferation in SW620 and SW480 cells. Control cells were transduced with empty lentiviral vector versus shRNA targeting
HMGA1. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; student’s t test). (B) Silencing HMGA1 decreases clonogenicity in SW620 and SW480
(**P < 0.01, ****P < 0.0001; student’s t test). (C) Predicted HMGA1 binding sites 1–5 and region 6–7 in the ASCL2 promoter region shown with
activating histone marks from SW620 and SW480 (GSE106921). (D) ChIP assay results at sites 1–5 and region 6–7 in SW620 cells from one repre-
sentative biological replicate for HMGA1 and activating histone marks (H3K4me3,and H3K27ac). (E) ChIP assay results at sites 1–5 and region 6–7 in
SW620 cells from one representative biological replicate for H3 and the repressive histone (H3K27me3). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P
< 0.0001; student’s t test following significance by ANOVA).

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J Clin Invest. 2025;135(3):e184442 https://doi.org/10.1172/JCI184442
We also identified inflammatory and proliferative networks
that are induced by HMGA1 and associated with HMGA1-depen-
dent accessible chromatin. Both IFN-α and γ signaling networks are
upregulated in crypt epithelial cells by HMGA1, leading to activa-
tion in IFN-stimulated genes and inflammatory networks, includ-
ing signal transduction and activator of transcription 1 and 2 (Stat1/2),
and chemokine genes encoding C-X-X motif chemokine ligands
9 and 10 (CXCL9/10). Importantly, inflammatory cytokines and/
or their receptors are often amenable to pharmacologic blockade.
Our scRNA-seq results show that tumor-infiltrating T-lymphocytes
increase in the setting of HMGA1 deficiency (Figure 7), suggesting
that HMGA1 in colon tumor cells may foster an immunologically
“cold” tumor microenvironment to facilitate tumor progression.
Alternatively, the changes in T cell number could reflect altered
transcriptomes from HMGA1 deficiency and associated changes
in cell behavior, including proliferation and motility. Given the
immune pathways identified from colon epithelial cells, studies
focusing on HMGA1 inflammatory networks and immune escape
are warranted and could identify new therapeutic strategies.
In summary, we discovered that HMGA1 acts as a molecular
key that “opens” chromatin to activate transcriptional networks that
maintain a stem and Paneth-like cell state early in colon tumori-
genesis. Within crypt epithelial cells, HMGA1 enhances chromatin
accessibility to activate the Ascl2 master regulator gene, additional
Wnt genes, and inflammatory networks in murine models with Apc
inactivation. Further, in human colon cancer, HMGA1 and ASCL2
are coexpressed and upregulated along with downstream Wnt path-
way genes. Together, our results establish HMGA1 as an epigenetic
gatekeeper of ASCL2 and Wnt signals, inflammation, and a stem-
like state in colon cells with APC inactivation, highlighting HMGA1
as a promising potential therapeutic target in colon cancer.
Methods
Sex as a biologic variable. All studies were carried out on male and female
mouse populations and similar findings were observed for both sexes.
Detailed methods, statistical analyses, and reagents are provided in
the supplemental material section, including culture medium, primers,
antibodies, and in silico approaches (Supplemental Table 5). Sequencing
data were deposited into the Gene Expression Omnibus (GSE) with acces-
sion numbers GSE279070 (scRNA-seq) and GSE278871 (ATAC-seq).
Animal models. CDX2P-CreERT2Apcfl/fl (73, 74) or ApcMin/– (Min mice)
(75–79) mice were previously described. The CDX2P-CreERT2Apcfl/fl were
generated and provided in house at the University of Michigan (73, 74).
The ApcMin/– (Min mice) were originally obtained from Bert Vogelstein at
Johns Hopkins University who developed this model (104). Both were
crossed to mice with global deficiency of one or both Hmga1 alleles (all
on C57Bl6 backgrounds) (28, 29). Tissue-specific Hmga1-deficient mod-
els were generated by crossing to mice with floxed Hmga1 alleles. Addi-
tional details are provided in the supplement (Supplemental Data Set 1).
Statistics. To compare continuous variables across 2 groups, statis-
tical significance was determined using a 2-tailed student’s t test when
normally distributed (ascertained by Ryan-Joyner and D’Agostino-Pear-
son tests). If not normal, the Mann-Whitney test was used. To compare
more than 2 groups, we used a 1-way ANOVA with Dunnett’s or Tur-
key’s multiple comparisons (Prism 10, GraphPad Software) after which 2
groups were compared via 2-tailed student’s t test if normally distributed
or Mann-Whitney if not. For categorical data, association with condition
Apc-deficient model led to the greatest impact on tumor develop-
ment and survival. However, in models of pancreatic tumorigen-
esis (28), we found a similar relationship whereby tissue-specific
loss of just one Hmga1 allele was sufficient to dampen tumori-
genesis and prolong survival, akin to results with tissue-specific
loss of both Hmga1 alleles. Intriguingly, crypt depth is similar
in the colon with intact Hmga1 or complete loss of Hmga1 in
the WT Apc epithelial compartment. Based on our transcriptom-
ic data showing that HMGA1 deficiency fosters differentiation
of stem cells to enterocytes, we surmise that the crypt depth is
maintained in the Hmga1 deficient model lacking Apc mutation
through skewing of more quiescent stem cells towards prolif-
erating enterocytes. Unfortunately, there are no pharmacologic
inhibitors to directly disrupt HMGA1 function in the clinics,
although modulating HMGA1 function or levels by approxi-
mately 50% is likely to be a more feasible therapeutic goal than
a more comprehensive disruption of its function.
To identify potential therapies to disrupt HMGA1 function,
we focused on the epigenetic landscape downstream of HMGA1.
HMGA1 enhances chromatin accessibility globally, in addition to
“opening” regulatory regions of the genome important for activation
of proliferation, inflammation, and Wnt signaling genes, including
Ascl2. ASCL2 functions as a master regulator of stemness in small
intestinal epithelium where it activates its own expression and that
of downstream Wnt effector and receptor genes (95–97), and in
esophageal cancer (98), although its role in colon epithelium had not
been studied in detail. A recent study also links ASCL2 expression to
early-onset colorectal cancer in a Japanese cohort (102).
We also found that HMGA1 binds directly to the ASCL2 pro-
moter region and recruits activating histones (H3K4me3, H3K27Ac)
to upregulate its expression. Indeed, Hmga1 and Ascl2 are the most
tightly coregulated genes in murine crypt epithelium (Figure 7).
Hmga1 is also coregulated with other Wnt effectors (Axin2, Tcf4,
Ctnnb1, cMyc, Sox9) and Wnt agonist receptors (Lgr5, Lrp6, Fzd7,
and Ephb2). In addition, HMGA1 enhances chromatin accessibil-
ity at promoter regions for Tcf4, Prom1, Lgr5, and Lrp5, suggesting
that it may directly induce these Wnt genes or modify chromatin to
facilitate their expression. In small intestinal stem cells and Caco2
cells (a human colon cancer cell line), HMGA1 directly induc-
es SOX9, and upregulation of SOX9 by HMGA1 could enhance
expansion in Paneth-like cells, since Paneth cell differentiation in
small intestine depends on SOX9 (30). While classical Paneth cells
are not present in the colon, Paneth cell metaplasia occurs in the
proximal colon in Apc mice (73, 74) and in humans with inflamma-
tory bowel disease (103), although their role in tumorigenesis is not
yet clear. HMGA1 also correlates with MYC in human colon cancer
and prior work in other settings shows that MYC directly induces
HMGA1 (70). HMGA1 also binds to the MYC promoter to induce
its expression in embryonic stem cells (34). In colon crypt cells,
HMGA1 enhances chromatin accessibility at the Hmga1 promoter,
suggesting that HMGA1 induces its own expression, a feature com-
mon to many stemness transcriptional regulators, such as ASCL2
(95). Given the link between ASCL2 and early onset colon cancer
(102), further studies to explore HMGA1 and ASCL2 are warranted.
Restoration of ASCL2 only partially rescues proliferation in colon
cancer cell lines with HMGA1 silencing, indicating that HMGA1
regulates additional networks during colon tumorigenesis.

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18
JHK, IH, SW, XW, SSP, JC, LC, LX, BEW, JCE, JK, ZT, IM,
KLR, YF, ERF, CLS, and LR performed experiments and ana-
lyzed data. TL interpreted histology.
Acknowledgments
This research was supported by the National Institutes of Health
(R01 CA293602, R01 CA232741, R01 HL145780, R01 DK 102943,
R01 HL143818), the Maryland Stem Cell Research Fund, and
National Cancer Center grants (Korea; NCC-2311410 and 2310390).
Address correspondence to: Linda Resar, Johns Hopkins Uni-
versity SOM, 720 Rutland Avenue, Baltimore, Maryland 21205,
USA. Phone: 410.614.0712; Email: lresar@jhmi.edu. Or to: Cyn-
thia Sears, Johns Hopkins University SOM, 1550 Orleans Street,
CRB2, Suite 1M.05, Baltimore, Maryland 21231, USA. Phone:
410.614.8378; Email: csears@jhmi.edu.
was evaluated by Fisher’s exact test. We compared survival analyses
under the assumption of Cox proportional hazards using the log-rank
test. P < 0.05 was considered significant. All code for the scRNA-seq
analysis was performed using Seurat at the indicated resolutions; code
will be made available from the corresponding author upon request.
Study approvals. All mouse studies were approved by the Johns Hop-
kins University Institutional Animal Care and Use Committee (IACUC).
Data availability. As above, metadata are available in the NCBI
GEO database (access numbers: scRNA-seq: GSE279070 and ATAC-
seq: GSE278871); the remaining data are provided in the Supporting
Data Values file.
Author contributions
LR and CLS conceptualized the project; LZL, IH, and BEW
drafted parts of the manuscript, and LR wrote the final draft,
which was reviewed by all authors prior to submission. LZL,
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