Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity

Abstract

The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value.

FIG 1

Characterization of STAT3-null MEFs stably expressing WT or NTD mutant STAT3. STAT3-null MEFs stably expressing WT STAT3 or STAT3 W37F, ΔNTD, or V77A/L78A were stimulated with LIF and analyzed by immunoblotting. (−), negative control for STAT3 expression from parental STAT3-null MEFs; unstim., unstimulated. Tubulin served as a loading control.

FIG 2

Optimization of the time point and cytokine concentration for mRNA and ChIP analyses. Wild-type MEFs and STAT3-null MEFs stably expressing WT STAT3 were stimulated with LIF (10 ng/ml) for different times (A) and with different concentrations of LIF for 30 min (B) and then analyzed by qRT-PCR for expression of the indicated STAT3 target genes. Data on the levels of expression were normalized to the level of HPRT expression and then to the level of mRNA expression in unstimulated cells.

FIG 3

NTD deletion reduces the level of induction of LIF-upregulated genes. (A) Induction of the top 100 LIF-upregulated genes and 100 non-LIF-regulated genes in STAT3-null MEFs stably expressing WT STAT3 or STAT3 ΔNTD. (B) Percentage of genes from panel A with significantly altered induction (20% threshold) in MEFs expressing STAT3 ΔNTD relative to that in MEFs expressing WT STAT3. (C) qRT-PCR validation of select LIF-upregulated genes in STAT3-null MEFs stably expressing WT STAT3 or STAT3 ΔNTD (the level of STAT3 expression was normalized to the level of HPRT expression).

FIG 4

NTD deletion reduces the induction of STAT3 target genes at low concentrations of LIF. (A) STAT3-null MEFs transiently transfected with WT STAT3 or STAT3 ΔNTD (0.2 μg/ml) for 24 h and then stimulated with the indicated concentrations of LIF for 15 min were analyzed by immunoblotting (actin served as a loading control). (B) STAT3-null MEFs transiently transfected with WT STAT3 or STAT3 ΔNTD and then stimulated with LIF (0.5 ng/ml) for 30 min were analyzed by qRT-PCR for expression of the indicated STAT3 target genes (the level of STAT3 expression was normalized to the level of HPRT expression; data are representative of those from 3 experiments).

FIG 5

NTD deletion reduces STAT3 DNA binding to target genes. (A) Regulatory regions of LIF-induced genes containing tandem STAT3-binding motifs (blue, strong binding sites; red, weak binding sites). The chromosome (chr) locations given are based on the version mm9 assembly. (B) STAT3-null MEFs stably expressing WT STAT3 or STAT3 ΔNTD were stimulated with LIF and then analyzed by chromatin immunoprecipitation with an antibody for STAT3, followed by qRT-PCR using primers flanking the STAT3-binding sites indicated in panel A. Data are expressed as the fold change in the level of expression as a percentage of the input in LIF-stimulated versus unstimulated cells (n = 3). (C) RNA-Seq transcript levels in STAT3-null MEFs stably expressing WT STAT3 or STAT3 ΔNTD. Data are expressed as the fold change in the level of mRNA expression in LIF-stimulated versus unstimulated cells.

FIG 6

Crystal structure of the STAT3 NTD. (A) Overall structure of the STAT3 NTD monomer at two viewing angles. (B) Two interfaces in the crystal structure are observed: a handshake dimer interface and an Ni²⁺-mediated tetramer interface. (C) The two molecules of the handshake dimer are related by a 2-fold noncrystallographic symmetry (NCS) axis. V77 and L78 dock into the opposing molecule in a cavity mainly created by the three N-terminal helices. Multiple hydrogen bonds also form on the dimer interface. (D) Another 2-fold NCS which involves multiple hydrogen bonds between two long helices antiparallel to each other from two NTD molecules is observed in the crystal. An Ni²⁺ ion sits in the middle of the axis and is coordinated by four H⁵⁸ residues, thus linking four handshake STAT3 dimers into an octamer. Also shown is the C terminus of each NTD which links to the STAT3 core domain.

FIG 7

Model of cooperative binding between two STAT3 dimers. (A) The asymmetric unit of the STAT3 NTD crystal contains five copies of the molecules. Along with one copy from the neighboring unit (E_sym), they form three handshake dimers with a similar organization, as shown from the superimposed image in panel B. (C) A model of two STAT3 dimers cooperatively binding to a tandem-site DNA with the help of NTD dimerization on each side.

FIG 8

Structural comparison and sequence alignment of the STAT3 NTD. (A) The NTDs of STAT1 (PDB accession number 1YVL), STAT3, and STAT4 (PDB accession number 1BGF) share similar handshake dimerization interfaces. The Ni²⁺ interface observed in STAT3 is similar to a second dimer interface observed in the crystal structure of the STAT4 NTD, and both interfaces contain W37 in the middle. The H⁵⁸ that coordinates Ni²⁺ in STAT3 corresponds to an Asn residue in STAT4. (B) Sequence alignment of NTD across STAT proteins. m, mouse; h, human. The red background indicates completely conserved residues; red text indicates partially conserved residues. Residue numbers of mouse STAT3 are at the top of the alignment. (C) The STAT3 NTD surface colored by conservation scores calculated by the ConSurf server based on the sequence alignment in panel B. The handshake interface is more conserved than the Ni²⁺ interface.

FIG 9

NTD mutations disrupt STAT3 cooperative DNA binding in vitro. (A) EMSA of purified P-STAT3 binding to an α2M DNA probe containing two STAT3-binding sites (5′-AGCAGTAACTGGAAAGTCCTTAATCCTTCTGGGAATTCT-3′; the STAT3-binding sites are underlined). The 5′ site is a weak binding site, while the 3′ site is a strong binding site. FL, full length. (B) EMSA of P-STAT3, STAT3 ΔNTD, or WT STAT3 with various DNA probes derived from the α2M promoter.

FIG 10

(A) Fluorescence polarization assay of WT and NTD mutant P-STAT3 binding to the indicated DNA probes. mP, millipolarization units. (B) Induction of SOCS3 mRNA levels (in LIF-stimulated versus unstimulated cells) in STAT3-null MEFs stably expressing WT STAT3 or STAT3 ΔNTD from RNA-Seq.

FIG 11

NTD interface mutations reduce induction of STAT3 target genes. (A) Induction of the top 100 LIF-upregulated genes in STAT3-null MEFs stably expressing WT STAT3 or NTD mutant STAT3. (B) Percentage of genes with significantly altered induction (20% threshold) in MEFs with STAT3 NTD mutants relative to that in MEFs with WT STAT3. (C) Overlap of genes affected by NTD point mutations versus the deletion mutation.

FIG 12

NTD interface mutations reduce induction of LIF-upregulated genes at low concentrations of LIF. (A) STAT3-null MEFs transiently transfected with WT STAT3 or NTD mutant STAT3 (0.2 μg/ml) for 24 h and then stimulated with LIF (0.5 ng/ml) were analyzed by immunoblotting (tubulin served as a loading control). (B) STAT3-null MEFs transiently transfected with WT STAT3 or NTD mutant STAT3 (0.2 μg/ml) for 24 h and then stimulated with the indicated concentrations of LIF were analyzed by qRT-PCR for STAT3 target gene expression (the level of expression was normalized to the level of HPRT expression; data are representative of those from 3 experiments).

FIG 13

Fold change of the top 100 LIF-downregulated genes in STAT3-null MEFs stably expressing WT STAT3 or NTD mutant STAT3.