Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells
Nucleic Acids Research
Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells
Zhongcheng Shi 2
Chi-I Chiang 2
Paul Labhart 1
Yanling Zhao 0
Jianhua Yang 0
Toni-Ann Mistretta 2
Susan J. Henning 4
Sankar N. Maity 3
Yuko Mori-Akiyama 2
0 Department of Pediatrics, Baylor College of Medicine, Texas Children's Cancer Center , Houston, TX 77030-2399 , USA
1 Active Motif , 1914 Palomar Oaks Way, Suite 150, Carlsbad, CA 92008 , USA
2 Department of Pathology & Immunology, Baylor College of Medicine, Texas Children's Hospital , 1102 Bates Street, Suite FC 830.27, Houston, TX 77030-2399 , USA
3 Department of Genitourinary Medical Oncology--Research, Division of Cancer Medicine, University of Texas M. D. Anderson Cancer Center , Houston, TX 77030 , USA
4 Department of Medicine, Cell Biology & Physiology, University of North Carolina at Chapel Hill , Chapel Hill, NC 27599-7032 , USA
Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.
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INTRODUCTION
SOX proteins, high-mobility group, (HMG)-box
transcription factors, play crucial roles in embryonic and adult
diverse tissues; these include maintenance of stem cell
properties, lineage specification and terminal differentiation in
a cell-type and tissue-specific manner. In the intestinal
epithelium, SOX9 is expressed in the stem/progenitor cells, as
well as in the nuclei of terminally differentiated Paneth cells
of the small intestinal crypts and tuft cells in the villi and it
plays a crucial role in Paneth cell differentiation (1,2).
Aberrant expression of SOX9 in some human cancers, including
colorectal cancer and in Apc(min/+) mouse intestinal
adenomas suggest roles of SOX9 in colorectal cancer and to
delineate the roles of SOX9 in colorectal cancer has been a
focus of some recent studies. However, there have been reports
of opposing SOX9 functions, promoting and suppressing of
proliferation, which suggest diverse, context-specific
functions of SOX9 in proliferation. This has been shown in
both developmental and cancer contexts. For instance, in
normal chondrocytic CFK2 cells and M12 prostate tumor
cells, SOX9 overexpression resulted in decreased
proliferation rate and cell cycle arrest in G0/G1 (3,4), while Sox9
knockdown in rat mesenchymal stem cells (MSCs) resulted
in a marked decrease in proliferation rate through delayed
S-phase progression and increased nuclear localization of
p21 (5). Furthermore, differential roles of SOX9 have been
demonstrated in normal intestinal epithelium; low SOX9
expression was associated with enhanced proliferative
capacity and high SOX9 expression suppressed proliferation
(6). Another study showed that SOX9 expression facilitated
growth and proliferation of colorectal cancer cells, whereas
inactivation reduced tumorigenicity (7).
To gain insight into SOX9-mediated transcriptional
regulation in colorectal cancer cells, we first attempted to
identify its physiological targets on a genome-scale
using chromatin immunoprecipitation (ChIP) followed by
sequencing (ChIP-seq) in human colorectal cancer cells. Our
ChIP-seq analysis revealed a large number of SOX9
transcriptional targets in diverse pathways. Interestingly,
motif analysis revealed CCAAT, a binding sequence for the
heterotrimeric NF-Y transcription factor, as a preferred
SOX9 binding sequence, in addition to the previously
identified classical consensus motif, A/TA/TCAAA/TG.
Statistical analysis of the ChIP-seq data further revealed that many
physiological SOX9 targets through the CCAAT motif are
on cell cycle regulatory genes, including the promoters of
well-characterized G2/M-specific genes, such as cyclin B1
(CCNB1) (8), cyclin B2 (CCNB2) (9), cyclin dependent
kinase 1 (CDK1) (10) and topoisomerase II (TOP2A) (11).
The current st (...truncated)